Markus Albert

An RLP23–SOBIR1–BAK1 complex mediates NLP-triggered immunity

Plants and animals employ innate immune systems to cope with microbial infection. Pattern-triggered immunity relies on the recognition of microbe-derived patterns by pattern recognition receptors (PRRs). Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) constitute plant immunogenic patterns that are unique, as these proteins are produced by multiple prokaryotic (bacterial) and eukaryotic (fungal, oomycete) species. Here we show that the leucine-rich repeat receptor protein (LRR-RP) RLP23 binds in vivo to a conserved 20-amino-acid fragment found in most NLPs (nlp20), thereby mediating immune activation in Arabidopsis thaliana. RLP23 forms a constitutive, ligand-independent complex with the LRR receptor kinase (LRR-RK) SOBIR1 (Suppressor of Brassinosteroid insensitive 1 (BRI1)-associated kinase (BAK1)-interacting receptor kinase 1), and recruits a second LRR-RK, BAK1, into a tripartite complex upon ligand binding. Stable, ectopic expression of RLP23 in potato (Solanum tuberosum) confers nlp20 pattern recognition and enhanced immunity to destructive oomycete and fungal plant pathogens, such as Phytophthora infestans and Sclerotinia sclerotiorum. PRRs that recognize widespread microbial patterns might be particularly suited for engineering immunity in crop plants.

The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium

A sulfated peptide activates a rice immune receptor. Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals.

Peptides as triggers of plant defence

Plants are confronted with several biotic stresses such as microbial pathogens and other herbivores. To defend against such attackers, plants possess an array of pattern recognition receptors (PRRs) that sense the danger and consequently initiate a defence programme that prevents further damage and spreading of the pest. Characteristic pathogenic structures, so-called microbe-associated molecular patterns (MAMPs), serve as signals that allow the plant to sense invaders. Additionally, pathogens wound or damage the plant and the resulting release of damage-associated molecular patterns (DAMPs) serves as a warning signal. This review focuses on peptides that serve as triggers or amplifiers of plant defence and thus follow the definition of a MAMP or a DAMP.

Tools and Strategies to Match Peptide-Ligand Receptor Pairs

Peptide signals have emerged as an important class of regulators in cell-to-cell communication in plants. Several families of small, secreted proteins with a conserved C-terminal Pro-rich motif have been identified as functional peptide signals in Arabidopsis thaliana. These proteins are presumed to be trimmed proteolytically and undergo posttranslational modifications, such as hydroxylation of Pro residues and glycosylation, to form mature, bioactive signals. Identification and matching of such ligands with their respective receptors remains a major challenge since the genes encoding them often show redundancy and low expression restricted to a few cells or particular developmental stages. To overcome these difficulties, we propose the use of ectopic expression of receptor genes in suitable plant cells like Nicotiana benthamiana for testing ligand candidates in receptor output assays and in binding studies. As an example, we used the IDA peptide HAE/HSL2 receptor signaling system known to regulate floral organ abscission. We demonstrate that the oxidative burst response can be employed as readout for receptor activation by synthetic peptides and that a new, highly sensitive, nonradioactive labeling approach can be used to reveal a direct correlation between peptide activity and receptor affinity. We suggest that these approaches will be of broad value for the field of ligand-receptor studies in plants.

Arabidopsis thaliana Pattern Recognition Receptors for Bacterial Elongation Factor Tu and Flagellin Can Be Combined to Form Functional Chimeric Receptors

The receptor kinase EFR of Arabidopsis thaliana detects the microbe-associated molecular pattern elf18, a peptide that represents the N terminus of bacterial elongation factor Tu. Here, we tested subdomains of EFR for their importance in receptor function. Transient expression of tagged versions of EFR and EFR lacking its cytoplasmic domain in leaves of Nicotiana benthamiana resulted in functional binding sites for elf18. No binding of ligand was found with the ectodomain lacking the transmembrane domain or with EFR lacking the first 5 of its 21 leucine-rich repeats (LRRs). EFR is structurally related to the receptor kinase flagellin-sensing 2 (FLS2) that detects bacterial flagellin. Chimeric receptors with subdomains of FLS2 substituting for corresponding parts of EFR were tested for functionality in ligand binding and receptor activation assays. Substituting the transmembrane domain and the cytoplasmic domain resulted in a fully functional receptor for elf18. Replacing also the outer juxtamembrane domain with that of FLS2 led to a receptor with full affinity for elf18 but with a lower efficiency in response activation. Extending the substitution to encompass also the last two of the LRRs abolished binding and receptor activation. Substitution of the N terminus by the first six LRRs from FLS2 reduced binding affinity and strongly affected receptor activation. In summary, chimeric receptors allow mapping of subdomains relevant for ligand binding and receptor activation. The results also show that modular assembly of chimeras from different receptors can be used to form functional receptors.

The Receptor-Like Protein ReMAX of Arabidopsis Detects the Microbe-Associated Molecular Pattern eMax from Xanthomonas

The receptor-like protein ReMAX/RLP1 of Arabidopsis functions as a pattern recognition receptor for eMax, a microbe-associated molecular pattern (MAMP) from Xanthomonas. Nicotiana benthamiana has no detection system for eMax but gains responsiveness to this MAMP when expressing a hybrid receptor with the extracellular domain of ReMAX and the cytoplasmic part of the related Eix2 from tomato. As part of their immune system, plants have pattern recognition receptors (PRRs) that can detect a broad range of microbe-associated molecular patterns (MAMPs). Here, we identified a PRR of Arabidopsis thaliana with specificity for the bacterial MAMP eMax from xanthomonads. Response to eMax seems to be restricted to the Brassicaceae family and also varied among different accessions of Arabidopsis. In crosses between sensitive accessions and the insensitive accession Shakhdara, eMax perception mapped to RECEPTOR-LIKE PROTEIN1 (RLP1). Functional complementation of rlp1 mutants required gene constructs that code for a longer version of RLP1 that we termed ReMAX (for receptor of eMax). ReMAX/RLP1 is a typical RLP with structural similarity to the tomato (Solanum lycopersicum) RLP Eix2, which detects fungal xylanase as a MAMP. Attempts to demonstrate receptor function by interfamily transfer of ReMAX to Nicotiana benthamiana were successful after using hybrid receptors with the C-terminal part of ReMAX replaced by that of Eix2. These results show that ReMAX determines specificity for eMax. They also demonstrate hybrid receptor technology as a promising tool to overcome problems that impede interfamily transfer of PRRs to enhance pathogen detection in crop plants.

Contamination Risks in Work with Synthetic Peptides: flg22 as an Example of a Pirate in Commercial Peptide Preparations

The pattern recognition receptor FLAGELLIN SENSING2 (FLS2) renders plant cells responsive to subnanomolar concentrations of flg22, the active epitope of bacterial flagellin. We recently observed that a preparation of the peptide IDL1, a signal known to regulate abscission processes via the receptor kinases HAESA and HAESA-like2, apparently triggered Arabidopsis thaliana cells in an FLS2-dependent manner. However, closer investigation revealed that this activity was due to contamination by a flg22-type peptide, and newly synthesized IDL1 peptide was completely inactive in FLS2 signaling. This raised alert over contamination events occurring in the process of synthesis or handling of peptides. Two recent reports have suggested that FLS2 has further specificities for structurally unrelated peptides derived from CLV3 and from Ax21. We thus scrutinized these peptides for activity in Arabidopsis cells as well. While responding to <1 nM flg22, Arabidopsis cells proved blind even to 100 μM concentrations of CLV3p and axYs22. Our results confirm the exquisite sensitivity and selectivity of FLS2 for flg22. They also show that inadvertent contaminations with flg22-type peptides do occur and can be detected even in trace amounts by FLS2.

Chimeric FLS2 Receptors Reveal the Basis for Differential Flagellin Perception in Arabidopsis and Tomato

Arabidopsis cells expressing either the FLS2 ortholog from Arabidopsis or tomato specifically respond to picomolar concentrations of the flg22 ligand. However, these orthologs exhibit characteristic differences when challenged with flg22 variants. Hybrid forms made from both orthologs allowed attribution of these differences to distinct subsets within the 28 Leucine-rich repeats of their ectodomains. The flagellin receptor of Arabidopsis thaliana, At-FLAGELLIN SENSING2 (FLS2), has become a model for mechanistic and functional studies on plant immune receptors. Here, we started out with a comparison of At-FLS2 and the orthologous tomato (Solanum lycopersicum) receptor Sl-FLS2. Both receptors specifically responded to picomolar concentrations of the genuine flg22 ligand but proved insensitive to >106-fold higher concentrations of CLV3 peptides that have recently been reported as a second type of ligand for At-FLS2. At-FLS2 and Sl-FLS2 exhibit species-specific differences in the recognition of shortened or sequence-modified flg22 ligands. To map the sites responsible for these species-specific traits on the FLS2 receptors, we performed domain swaps, substituting subsets of the 28 leucine-rich repeats (LRRs) in At-FLS2 with the corresponding LRRs from Sl-FLS2. We found that the LRRs 7 to 10 of Sl-FLS2 determine the high affinity of Sl-FLS2 for the core part RINSAKDD of flg22. In addition, we discovered importance of the LRRs 19 to 24 for the responsiveness to C-terminally modified flagellin peptides. These results indicate that ligand perception in FLS2 is a complex molecular process that involves LRRs from both the outermost and innermost LRRs of the FLS2 ectodomain.

Regulation of cell behaviour by plant receptor kinases: Pattern recognition receptors as prototypical models.

In this review we focus on pattern recognition receptors in plants that detect extracellular signals indicative for pathogen attack and injury. We start out with a discussion on FLS2, which binds and responds to bacterial flagellin, and then concentrate on ligand-receptor interactions as initial steps in the molecular receptor activation process. Comparison with other receptor kinases, whether involved in plant immunity or regulation of other cellular programs, might indicate common principles of receptor activation.

Detection of the plant parasite Cuscuta reflexa by a tomato cell surface receptor.

Resistance is not, after all, futile The parasitic plant known as dodder attaches to its hosts and sucks the life out of them. Oddly, the common tomato stands tall when under attack. Hegenauer et al. have leveraged that difference to identify part of the molecular defense system that protects tomato plants (see the Perspective by Ntoukakis and Gimenez-Ibanez). In a process analogous to defenses mounted against microbial infection, the host plant perceives a small-peptide signal from the parasitic plant and initiates defense responses. The candidate receptor isolated from the tomato plant provided partial protection when transferred to two other susceptible plant species. Science, this issue p. 478; see also p. 442 Tomato plants rebuff attack by a parasitic plant upon perceiving a peptide signal from the invader. Parasitic plants are a constraint on agriculture worldwide. Cuscuta reflexa is a stem holoparasite that infests most dicotyledonous plants. One exception is tomato, which is resistant to C. reflexa. We discovered that tomato responds to a small peptide factor occurring in Cuscuta spp. with immune responses typically activated after perception of microbe-associated molecular patterns. We identified the cell surface receptor-like protein CUSCUTA RECEPTOR 1 (CuRe1) as essential for the perception of this parasite-associated molecular pattern. CuRe1 is sufficient to confer responsiveness to the Cuscuta factor and increased resistance to parasitic C. reflexa when heterologously expressed in otherwise susceptible host plants. Our findings reveal that plants recognize parasitic plants in a manner similar to perception of microbial pathogens.