Plant-Microbe Symbiosis

During root nodule symbiosis (RNS), cell-division activity is re-initiated and sustained in the root cortex to create a hospitable cellular niche. Such temporary and spatially confined site is required to render host cells compatible with the intracellular progression of rhizobia. Although it has been suggested that early infection events might involve a pre-mitotic cell-cycle arrest, this process has not been dissected with cellular resolution. Here, we show that a dual-colour Medicago histone reporter robustly identifies cells with different mitotic or endoreduplication activities in the root cortex. By imaging deep root tissues, we found that a confined trajectory of cortical cells that are transcellularly passed by infection threads are in a stage of the cell-cycle that is distinct from directly adjacent cells. Distinctive features of infected cells include nuclear widening and large-scale chromatin rearrangements consistent with a cell-cycle exit prior to differentiation. Using a combination of fluorescent reporters demarcating cell-cycle phase progression, we confirmed that a reduced proliferation potential and modulating the G2/M transition, a process possibly controlled by the NF-YA1 transcription factor, mark the success of rhizobial delivery to nodule cells.

Plant adaptation to drought facilitates major ecological transitions, and will likely play a vital role under looming climate change. Mycorrhizas, i.e. strategic associations between plant roots and soil-borne symbiotic fungi, can exert strong influence on the tolerance to drought of extant plants. Here, I show how mycorrhizal strategy and drought adaptation have been shaping one another throughout the course of plant evolution. To characterize the evolutions of both plant characters, I applied a phylogenetic comparative method using data of 1,638 extant species globally distributed. The detected correlated evolution unveiled gains and losses of drought tolerance occurring at faster rates in lineages with ecto- or ericoid mycorrhizas, which were on average about 15 and 300 times faster than in lineages with the arbuscular mycorrhizal and naked root (non-mycorrhizal alone or with facultatively arbuscular mycorrhizal) strategy, respectively. My study suggests that mycorrhizas can play a key facilitator role in the evolutionary processes of plant adaptation to critical changes in water availability across global climates.

Cu+-chaperones are a diverse group of proteins that allocate Cu+ ions to specific copper-proteins, creating different copper pools targeted to specific physiological processes. Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper e.g. for energy delivery via respiration, for which targeted copper deliver systems would be required. MtNCC1 is a nodule-specific Cu+-chaperone encoded in the Medicago truncatula genome, with a N-terminus Atx1-like domain that can bind Cu+ with picomolar affinities. This gene is expressed primarily from the late infection zone to the early fixation zone, and is located in the cytosol, associated to plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation, ncc1 mutants have a severe reduction of nitrogenase activity, and a 50% reduction in copper-dependent cytochrome c oxidase activity. A subset of the copper-proteome is also affected in the mutant nodules. Many of these proteins can be pulled-down when using a Cu+-loaded N-terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper-dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.

Bacterial surface motility is a complex microbial trait that contributes to host colonization. However, the knowledge about regulatory mechanisms that control surface translocation in rhizobia and their role in the establishment of symbiosis with legumes is still limited. Recently, 2-tridecanone (2-TDC) was identified as an infochemical in bacteria that hampers microbial colonization of plants. In the alfalfa symbiont Sinorhizobium meliloti, 2-TDC promotes a mode of surface motility that is mostly independent of flagella. To understand the mechanism of action of 2-TDC in S. meliloti and unveil genes putatively involved in plant colonization, Tn5 transposants derived from a flagellaless strain that were impaired in 2-TDC-induced surface spreading were isolated and genetically characterized. In one of the mutants, the gene coding for the chaperone DnaJ was inactivated. Characterization of this transposant and newly obtained flagella-minus and flagella-plus dnaJ deletion mutants revealed that DnaJ is essential for surface translocation, while it plays a minor role in swimming motility. DnaJ loss-of-function reduces salt and oxidative stress tolerance in S. meliloti and hinders the establishment of efficient symbiosis by affecting nodule formation efficiency, cellular infection, and nitrogen fixation. Intriguingly, the lack of DnaJ causes more severe defects in a flagellaless background. This work highlights the role of DnaJ in the free-living and symbiotic lifestyles of S. meliloti.

Fungal-bacterial symbioses range from antagonisms to mutualisms and remain one of the least understood interdomain interactions despite their ubiquity as well as ecological and medical importance. To build a predictive conceptual framework for understanding interactions between fungi and bacteria in different types of symbioses, we surveyed fungal and bacterial transcriptional responses in the mutualism between Rhizopus microsporus (Rm) (ATCC 52813, host) and its Mycetohabitans (formerly Burkholderia) endobacteria versus the antagonism between a nonhost Rm (ATCC 11559) and Mycetohabitans isolated from the host, at two time points, before and after partner physical contact. We found that bacteria and fungi sensed each other before contact and altered gene expression patterns accordingly. Mycetohabitans did not discriminate between the host and nonhost and engaged a common set of genes encoding known as well as novel symbiosis factors. In contrast, responses of the host versus nonhost to endobacteria were dramatically different, converging on the altered expression of genes involved in cell wall biosynthesis and reactive oxygen species (ROS) metabolism. On the basis of the observed patterns, we formulated a set of hypotheses describing fungal-bacterial interactions and tested some of them. By conducting ROS measurements, we confirmed that nonhost fungi increased production of ROS in response to endobacteria, whereas host fungi quenched their ROS output, suggesting that ROS metabolism contributes to the nonhost resistance to bacterial infection and the host ability to form a mutualism. Overall, our study offers a testable framework of predictions describing interactions of early divergent Mucoromycotina fungi with bacteria.

Symbiosis between leguminous plants and soil bacteria rhizobia is a refined type of plant–microbial interaction that has a great importance to the global balance of nitrogen. The reduction of atmospheric nitrogen takes place in infected cells of a root nodule that serves as a temporary shelter for thousands of living bacteria, which, per se, is an unusual state of a eukaryotic cell. One of the most striking features of an infected cell is the drastic changes in the endomembrane system that occur after the entrance of bacteria to the host cell symplast. Mechanisms for maintaining intracellular bacterial colony represent an important part of symbiosis that have still not been sufficiently clarified. This review focuses on the changes that occur in an endomembrane system of infected cells and on the putative mechanisms of infected cell adaptation to its unusual lifestyle.

• Fitness of rhizobia is a product of their competitive success in diverse niches.

• Competition can occur within and between species, and for different resources.

• Competition can be modulated by abiotic factors and by legume hosts.

• New techniques are revealing diverse mechanisms of competition.

• Competitive mechanisms in bulk soil and nodule senescence remain underexplored.

Symbioses with beneficial microbes are widespread in plants, but these relationships must balance the energy invested by the plants with the nutrients acquired. Symbiosis with arbuscular mycorrhizal (AM) fungi occurs throughout land plants but our understanding of the genes and signals that regulate colonisation levels is limited. Here, we demonstrate that in tomato two CLV3/EMBRYO-SURROUNDING REGION (CLE) peptides, SlCLE10 and SlCLE11, act to suppress AM colonisation of roots. Mutant studies and overexpression via hairy transformation indicate SlCLE11 acts locally in the root to limit AM colonisation. Indeed, SlCLE11 expression is strongly induced in AM colonised roots but SlCLE11 is not required for phosphate suppression of AM colonisation. SlCLE11 may act through as yet uncharacterised signalling pathways, as SlCLE11 does not suppress AM colonisation by acting through two previously characterised receptors with roles in regulating AM colonisation, SlFAB (CLAVATA1 orthologue) or SlCLV2. SlCLE10 appears to play a more minor or redundant role, as cle10 mutants did not influence AM, although the fact that ectopic overexpression of SlCLE10 did suppress colonisation suggests SlCLE10 may play a role in regulating AM colonisation. Our findings show that CLE peptides regulate AM colonisation in the non-legume species tomato.

Background
The nitrate regulates soybean nodulation and nitrogen fixation systemically, mainly in inhibiting nodule growth and reducing nodule nitrogenase activity, but the reason for its inhibition is still inconclusive.

Methods
The systemic effect of nitrate on nodule structure, function, and carbon distribution in soybean (Glycine max (L.) Merr.) was studied in a dual-root growth system, with both sides inoculated with rhizobia and only one side subjected to nitrate treatment for four days. The non-nodulating side was genetically devoid of the ability to form nodules. Nutrient solutions with nitrogen concentrations of 0, 100, and 200 mg L-1 were applied as KNO3 to the non-nodulating side, while the nodulating side received a nitrogen-free nutrient solution. Carbon partitioning in roots and nodules was monitored using 13C-labelled CO2. Other nodule responses were measured via the estimation of the nitrogenase activity and the microscopic observation of nodule ultrastructure.

Results
Elevated concentrations of nitrate applied on the non-nodulating side caused a decrease in the number of bacteroids, fusion of symbiosomes, enlargement of the peribacteroid spaces, and onset of degradation of poly-β-hydroxybutyrate granules, which is a form of carbon storage in bacteroids. These microscopic observations were associated with a strong decrease in the nitrogenase activity of nodules. Furthermore, our data demonstrate that the assimilated carbon is more likely to be allocated to the non-nodulating roots, as follows from the competition for carbon between the symbiotic and non-symbiotic sides of the dual-root system.

Conclusion
We propose that there is no carbon competition between roots and nodules when they are indirectly supplied with nitrate, and that the reduction of carbon fluxes to nodules and roots on the nodulating side is the mechanism by which the plant systemically suppresses nodulation under nitrogen-replete conditions.

Nitrogen is a major element for plant life, yet environmental nitrogen is poorly available to plant, and thus classified as a ‘limiting element’. As a consequence, most plants, except the insectivorous florae, rely upon microbial partners to maintain the nitrogen supply. Nitrogen-fixing prokaryotes are widely distributed and account for the fixation of nearly 50–200 megatonnes of nitrogen per year. Nitrogen-fixing microorganisms are potent agents for applications in agricultural fields. Reduction of gaseous dinitrogen to bioavailable nitrogen is mainly done by the molybdenum-dependent nitrogenase in archaea and eubacteria. In plants, the process of nodulation has evolved from 100 million years ago, confering the nodulation capability to about 70% of leguminous plant species. The genes necessary for the nitrogen fixation evolved only after the divergence of bacteria and archaea. Furthermore, the nitrogen-fixing endosymbionts are supposed to have evolved many times in the higher plants, especially in angiosperms. This chapter reviews the diversity and evolution of nitrogen-fixing bacteria.

Drought is one of the greatest threats to agricultural production. Legumes are relatively susceptible to drought and have the unique ability to establish symbiosis with nitrogen-fixing bacteria. Deciphering how symbiotic interactions affect legume stress tolerance is a relevant question that remains unsolved, especially in the context of maintaining crop production under climate change. Our study addresses the comparative analysis of drought response and drought tolerance in symbiosis-dependent versus N-fed plants. Our results support the existence of attenuation in the damage promoted by drought in nodulated pea and vetch plants. This increase in drought tolerance correlates with changes in physiological parameters, such the hydric status of the plant, changes in biochemical parameters, such as the accumulation of osmolytes and antioxidant pigments, or the protection against lipid peroxidation. We have also found changes at the molecular level, identifying different genomic pathways through transcriptomic analysis. Although this characterization strengthens the evidence for a common strategy of coping with water deprivation in nodulated and N-fed plants, the above-mentioned changes suggest divergent and specific strategies of drought tolerance in nodulated plants as well as differences between pea and common vetch. In addition, our transcriptomic studies help unravel the molecular mechanisms through which rhizobial symbiosis contributes to drought tolerance in Vicia sativa and Pisum sativum. These results highlight the potential of using effective Rhizobium inoculants as a tool to cope with drought and enhance N-fixing ability under agriculturally relevant conditions.

Ustilago maydis is a pathogenic fungus in Basidiomycota causing corn smut disease. A strain of U. maydis YZZF202006 was isolated from the tumor of corn smut collected from Jingzhou city in China. The intracellular bacteria were confirmed inner hyphal of the strain YZZF202006 by PCR amplification and fluorescence in situ hybridization (FISH) and SYTO-9. An endohyphal bacterium YZUMF202001 was isolated from the protoplasts of the strain YZZF202006. It was gram-negative, short rod-shaped with smooth light yellow colony. The endohyphal bacterium was genomic evidenced as Klebsiella michiganensis on the basis of average nucleotide identity (ANI) analysis and the phylogram. And then K. michiganensis was GFP-Labeled and reintroduced into U. maydis, which confirmed the bacterium can live in hpyphal of U.maydis. The bacterium can grow on N-free culture media. Its nitrogenase activity was reached av. 646.25 ± 38.61 nmol·mL−1·h−1 C2H4 by acetylene reduction assay. A cluster of nitrogen fixation genes (nifJHDKTXENXUSVWZMFLABQ) was found from its genome. The endohyphal K. michiganensis may play an important role to help nitrogen fixation for fungi in the future.

Understanding the interactions between plants and microorganisms can inform microbiome management to enhance crop productivity and resilience to stress. Here, we apply a genome-centric approach to identify ecologically important leaf microbiome members on replicated plots of field-grown switchgrass and miscanthus, and to quantify their activities over two growing seasons for switchgrass. We use metagenome and metatranscriptome sequencing and curate 40 medium- and high-quality metagenome-assembled-genomes (MAGs). We find that classes represented by these MAGs (Actinomycetia, Alpha- and Gamma- Proteobacteria, and Bacteroidota) are active in the late season, and upregulate transcripts for short-chain dehydrogenase, molybdopterin oxidoreductase, and polyketide cyclase. Stress-associated pathways are expressed for most MAGs, suggesting engagement with the host environment. We also detect seasonally activated biosynthetic pathways for terpenes and various non-ribosomal peptide pathways that are poorly annotated. Our findings support that leaf-associated bacterial populations are seasonally dynamic and responsive to host cues.

Legume-rhizobia nodulation is a complex developmental process. It initiates when the rhizobia-produced Nod factors are perceived by specific LysM receptors present in the root hair apical membrane. Consequently, SYMRK (Symbiosis Receptor-like Kinase) becomes active in the root hair and triggers an extensive signaling network essential for the infection process and nodule organogenesis. Despite its relevant functions, the underlying cellular mechanisms involved in SYMRK signaling activity remain poorly characterized. In this study, we demonstrated that PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis. We found that in uninoculated roots, PvSYMRK-EGFP is mainly associated with the plasma membrane, although intracellular puncta labelled with PvSymRK-EGFP were also observed in root hair and nonhair-epidermal cells. Inoculation with Rhizobium etli producing Nod factors induces in the root hair a redistribution of PvSYMRK-EGFP from the plasma membrane to intracellular puncta. In accordance, deletion of the endocytic motif YXXØ (YKTL) and treatment with the endocytosis inhibitors ikarugamycin (IKA) and tyrphostin A23 (TyrA23), as well as brefeldin A (BFA), drastically reduced the density of intracellular PvSYMRK-EGFP puncta. A similar effect was observed in the phosphorylation-deficient (T589A) and kinase-dead (K618E) mutants of PvSYMRK-EGFP, implying these structural features are positive regulators of PvSYMRK-EGFP endocytosis. Our findings lead us to postulate that rhizobia-induced endocytosis of SYMRK modulates the duration and amplitude of the SYMRK-dependent signaling pathway.

Legumes coordinate nodulation and plant development to maximize reproductive success, but the underlying molecular mechanisms are not well understood. A recent study by Yun et al. has revealed that nodulation drives root-to-shoot movement of microRNA172 (miR172) to accelerate flowering time, thus building a new bridge between nodulation and plant growth regulation.

Nitrogenase in some bacteria and archaea catalyzes conversion of N2 to ammonia. To reconstitute a nitrogenase biosynthetic pathway in a eukaryotic host is still a challenge, since synthesis of nitrogenase requires a large number of nif (nitrogen fixation) genes. Viral 2A peptide mediated “cleavage” of polyprotein is one of strategies for multigene co-expression. Here, we show that cleavage efficiency of NifB-2A-NifH polyprotein linked by four different 2A peptides (P2A, T2A, E2A, and F2A) in Saccharomyces cerevisiae ranges from ~50% to ~90%. The presence of a 2A tail in NifB, NifH, and NifD does not affect their activity. Western blotting shows that 9 Nif proteins (NifB, NifH, NifD, NifK, NifE, NifN, NifX, HesA, and NifV) from Paenibacillus polymyxa that are fused into two polyproteins via 2A peptides are co-expressed in S. cerevisiae. Expressed NifH from Klebsiella oxytoca NifU and NifS and P. polymyxa NifH fusion linked via 2A in S. cerevisiae exhibits Fe protein activity.

In plant–microbe interactions, symbionts and pathogens live within plants and attempt to avoid triggering plant defense responses. In order to do so, these microbes have evolved multiple mechanisms that target components of the plant cell nucleus. Rhizobia-induced symbiotic signaling requires the function of specific legume nucleoporins within the nuclear pore complex. Symbiont and pathogen effectors harbor nuclear localization sequences that facilitate movement across nuclear pores, allowing these proteins to target transcription factors that function in defense. Oomycete pathogens introduce proteins that interact with plant pre-mRNA splicing components in order to alter host splicing of defense-related transcripts. Together, these functions indicate that the nucleus is an active site of symbiotic and pathogenic functioning in plant–microbe interactions.

Biological nitrogen (N) fixation (BNF) via diazotrophs is an important ecological process for the conversion of atmospheric N to biologically available N. Although soil diazotrophs play a dominant role in BNF and arbuscular mycorrhizal fungi (AMF) serve as helpers to favor BNF, the response of soil BNF and diazotrophic communities to different long-term fertilizations and the role of AMF in diazotrophs-driven BNF are poorly understood. Herein, a 33-year fertilization experiment in a wheat–maize intercropping system was conducted to investigate the changes in soil BNF rates, diazotrophic and AMF communities, and their interactions after long-term representative fertilization (chemical fertilizer, cow manure, wheat straw, and green manure). We found a remarkable increase in soil BNF rates after more than three decades of fertilization compared with nonfertilized soil, and the green manure treatment rendered the highest enhancement. The functionality strengthening was mainly associated with the increase in the absolute abundance of diazotrophs and AMF and the relative abundance of the key ecological cluster of Module #0 (gained from the co-occurrence network of diazotrophic and AMF species) with dominant diazotrophs such as Skermanella and Azospirillum. Furthermore, although the positive correlations between diazotrophs and AMF were reduced under long-term organic fertilization regimes, green manuring could reverse the decline within Module #0, and this had a positive relationship with the BNF rate. This study suggests that long-term fertilization could promote N fixation and select specific groups of N fixers and their helpers in certain areas. Our work provides solid evidence that N fixation and certain groups of diazotrophic and AMF taxa and their interspecies relationship will be largely favored after the fertilized strategy of green manure.

Cover crops (CCs) were found to improve soil health by increasing plant diversity and ground cover. They may also improve water supply for cash crops by reducing evaporation and increasing soil water storage capacity. However, their influence on plant-associated microbial communities, including symbiotic arbuscular mycorrhizal fungi (AMF), is less well understood. In a corn field trial, we studied the response of AMF to a four-species winter CC, relative to a no-CC control, as well as to two contrasting water supply levels (i.e., drought and irrigated). We measured AMF colonization of corn roots and used Illumina MiSeq sequencing to study the composition and diversity of soil AMF communities at two depths (i.e., 0–10 and 10–20 cm). In this trial, AMF colonization was high (61–97%), and soil AMF communities were represented by 249 amplicon sequence variants (ASVs) belonging to 5 genera and 33 virtual taxa. Glomus, followed by Claroideoglomus and Diversispora (class Glomeromycetes), were the dominant genera. Our results showed interacting effects between CC treatments and water supply levels for most of the measured variables. The percentage of AMF colonization, arbuscules, and vesicles tended to be lower in irrigated than drought sites, with significant differences detected only under no-CC. Similarly, soil AMF phylogenetic composition was affected by water supply only in the no-CC treatment. Changes in the abundance of individual virtual taxa also showed strong interacting effects between CCs, irrigation, and sometimes soil depth, although CC effects were clearer than irrigation effects. An exception to these interactions was soil AMF evenness, which was higher in CC than no-CC, and higher under drought than irrigation. Soil AMF richness was not affected by the applied treatments. Our results suggest that CCs can affect the structure of soil AMF communities and modulate their response to water availability levels, although soil heterogeneity could influence the final outcome.

Plants have to cope with a myriad of soilborne pathogens that affect crop production and food security. The complex interactions between the root system and microorganisms are determinant for the whole plant health. However, the knowledge regarding root defense responses is limited as compared to the aerial parts of the plant. Immune responses in roots appear to be tissue-specific suggesting a compartmentalization of defense mechanisms in these organs. The root cap releases cells termed root “associated cap-derived cells” (AC-DCs) or “border cells” embedded in a thick mucilage layer forming the root extracellular trap (RET) dedicated to root protection against soilborne pathogens. Pea (Pisum sativum) is the plant model used to characterize the composition of the RET and to unravel its function in root defense. The objective of this paper is to review modes of action of the RET from pea against diverse pathogens with a special focus on root rot disease caused by Aphanomyces euteiches, one of the most widely occurring and large-scale pea crop diseases. The RET, at the interface between the soil and the root, is enriched in antimicrobial compounds including defense-related proteins, secondary metabolites, and glycan-containing molecules. More especially arabinogalactan proteins (AGPs), a family of plant extracellular proteoglycans belonging to the hydroxyproline-rich glycoproteins were found to be particularly present in pea border cells and mucilage. Herein, we discuss the role of RET and AGPs in the interaction between roots and microorganisms and future potential developments for pea crop protection.

Arbuscular mycorrhizal (AM) symbiosis is a wide-spread, ancient mutualistic association between plants and fungi, and facilitates nutrient uptake into plants. Cell surface receptor-like kinases (RLKs) and receptor-like cytoplasmic kinases (RLCKs) play pivotal roles in transmembrane signaling, while few RLCKs are known to function in AM symbiosis. Here, we show that 27 out of 40 AM-induced kinases (AMKs) are transcriptionally upregulated by key AM transcription factors in Lotus japonicus. Nine AMKs are only conserved in AM-host lineages, among which the SPARK-RLK-encoding gene KINASE3 (KIN3) and the RLCK paralogues AMK8 and AMK24 are required for AM symbiosis. KIN3 expression is directly regulated by the AP2 transcription factor CTTC MOTIF-BINDING TRANSCRIPTION FACTOR1 (CBX1), which regulates the reciprocal exchange of nutrients in AM symbiosis, via the AW-box motif in the KIN3 promoter. Loss of function mutations in KIN3, AMK8 or AMK24 result in reduced mycorrhizal colonization in L. japonicus. AMK8 and AMK24 physically interact with KIN3. KIN3 and AMK24 are active kinases and AMK24 directly phosphorylates KIN3 in vitro. Moreover, CRISPR-Cas9-mediated mutagenesis of OsRLCK171, the sole homolog of AMK8 and AMK24 in rice (Oryza sativa), leads to diminished mycorrhization with stunted arbuscules. Overall, our results reveal a crucial role of the CBX1-driven RLK/RLCK complex in the evolutionarily conserved signaling pathway enabling arbuscule formation.

Bacterial flagellin protein is a potent microbe-associated molecular pattern. Immune responses are triggered by a 22-amino-acid epitope derived from flagellin, known as flg22, upon detection by the pattern recognition receptor FLAGELLIN-SENSING2 (FLS2) in multiple plant species. However, increasing evidence suggests that flg22 epitopes of several bacterial species are not universally immunogenic to plants. We investigated whether flg22 immunogenicity systematically differs between classes of the phylum Proteobacteria, using a dataset of 2,470 flg22 sequences. To predict which species encode highly immunogenic flg22 epitopes, we queried a custom motif (11[ST]xx[DN][DN]xAGxxI21) in the flg22 sequences, followed by sequence conservation analysis and protein structural modeling. These data led us to hypothesize that most flg22 epitopes of the γ- and β-Proteobacteria are highly immunogenic, whereas most flg22 epitopes of the α-, δ-, and ε-Proteobacteria are weakly to moderately immunogenic. To test this hypothesis, we generated synthetic peptides representative of the flg22 epitopes of each proteobacterial class, and we monitored their ability to elicit an immune response in Arabidopsis thaliana. The flg22 peptides of γ- and β-Proteobacteria triggered strong oxidative bursts, whereas peptides from the ε-, δ-, and α-Proteobacteria triggered moderate, weak, or no response, respectively. These data suggest flg22 immunogenicity is not highly conserved across the phylum Proteobacteria. We postulate that sequence divergence of each taxonomic class was present prior to the evolution of FLS2, and that the ligand specificity of A. thaliana FLS2 was driven by the flg22 epitopes of the γ- and β-Proteobacteria, a monophyletic group containing many common phytopathogens.