Cyril Zipfel

Bacterial disease resistance in Arabidopsis through flagellin perception

Plants and animals recognize microbial invaders by detecting pathogen-associated molecular patterns (PAMPs) such as flagellin. However, the importance of flagellin perception for disease resistance has, until now, not been demonstrated. Here we show that treatment of plants with flg22, a peptide representing the elicitor-active epitope of flagellin, induces the expression of numerous defence-related genes and triggers resistance to pathogenic bacteria in wild-type plants, but not in plants carrying mutations in the flagellin receptor gene FLS2. This induced resistance seems to be independent of salicylic acid, jasmonic acid and ethylene signalling. Wild-type and fls2 mutants both display enhanced resistance when treated with crude bacterial extracts, even devoid of elicitor-active flagellin, indicating the existence of functional perception systems for PAMPs other than flagellin. Although fls2 mutant plants are as susceptible as the wild type when bacteria are infiltrated into leaves, they are more susceptible to the pathogen Pseudomonas syringae pv. tomato DC3000 when it is sprayed on the leaf surface. Thus, flagellin perception restricts bacterial invasion, probably at an early step, and contributes to the plants disease resistance.

Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation.

Higher eukaryotes sense microbes through the perception of pathogen-associated molecular patterns (PAMPs). Arabidopsis plants detect a variety of PAMPs including conserved domains of bacterial flagellin and of bacterial EF-Tu. Here, we show that flagellin and EF-Tu activate a common set of signaling events and defense responses but without clear synergistic effects. Treatment with either PAMP results in increased binding sites for both PAMPs. We used this finding in a targeted reverse-genetic approach to identify a receptor kinase essential for EF-Tu perception, which we called EFR. Nicotiana benthamiana, a plant unable to perceive EF-Tu, acquires EF-Tu binding sites and responsiveness upon transient expression of EFR. Arabidopsis efr mutants show enhanced susceptibility to the bacterium Agrobacterium tumefaciens, as revealed by a higher efficiency of T-DNA transformation. These results demonstrate that EFR is the EF-Tu receptor and that plant defense responses induced by PAMPs such as EF-Tu reduce transformation by Agrobacterium.

A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence

Plants sense potential microbial invaders by using pattern-recognition receptors to recognize pathogen-associated molecular patterns (PAMPs). In Arabidopsis thaliana, the leucine-rich repeat receptor kinases flagellin-sensitive 2 (FLS2) (ref. 2) and elongation factor Tu receptor (EFR) (ref. 3) act as pattern-recognition receptors for the bacterial PAMPs flagellin and elongation factor Tu (EF-Tu) (ref. 5) and contribute to resistance against bacterial pathogens. Little is known about the molecular mechanisms that link receptor activation to intracellular signal transduction. Here we show that BAK1 (BRI1-associated receptor kinase 1), a leucine-rich repeat receptor-like kinase that has been reported to regulate the brassinosteroid receptor BRI1 (refs 6,7), is involved in signalling by FLS2 and EFR. Plants carrying bak1 mutations show normal flagellin binding but abnormal early and late flagellin-triggered responses, indicating that BAK1 acts as a positive regulator in signalling. The bak1-mutant plants also show a reduction in early, but not late, EF-Tu-triggered responses. The decrease in responses to PAMPs is not due to reduced sensitivity to brassinosteroids. We provide evidence that FLS2 and BAK1 form a complex in vivo, in a specific ligand-dependent manner, within the first minutes of stimulation with flagellin. Thus, BAK1 is not only associated with developmental regulation through the plant hormone receptor BRI1 (refs 6,7), but also has a functional role in PRR-dependent signalling, which initiates innate immunity.

The N Terminus of Bacterial Elongation Factor Tu Elicits Innate Immunity in Arabidopsis Plants

Innate immunity is based on the recognition of pathogen-associated molecular patterns (PAMPs). Here, we show that elongation factor Tu (EF-Tu), the most abundant bacterial protein, acts as a PAMP in Arabidopsis thaliana and other Brassicaceae. EF-Tu is highly conserved in all bacteria and is known to be N-acetylated in Escherichia coli. Arabidopsis plants specifically recognize the N terminus of the protein, and an N-acetylated peptide comprising the first 18 amino acids, termed elf18, is fully active as inducer of defense responses. The shorter peptide, elf12, comprising the acetyl group and the first 12 N-terminal amino acids, is inactive as elicitor but acts as a specific antagonist for EF-Tu–related elicitors. In leaves of Arabidopsis plants, elf18 induces an oxidative burst and biosynthesis of ethylene, and it triggers resistance to subsequent infection with pathogenic bacteria.

Transgeneration memory of stress in plants

Owing to their sessile nature, plants are constantly exposed to a multitude of environmental stresses to which they react with a battery of responses. The result is plant tolerance to conditions such as excessive or inadequate light, water, salt and temperature, and resistance to pathogens. Not only is plant physiology known to change under abiotic or biotic stress, but changes in the genome have also been identified. However, it was not determined whether plants from successive generations of the original, stressed plants inherited the capacity for genomic change. Here we show that in Arabidopsis thaliana plants treated with short-wavelength radiation (ultraviolet-C) or flagellin (an elicitor of plant defences), somatic homologous recombination of a transgenic reporter is increased in the treated population and these increased levels of homologous recombination persist in the subsequent, untreated generations. The epigenetic trait of enhanced homologous recombination could be transmitted through both the maternal and the paternal crossing partner, and proved to be dominant. The increase of the hyper-recombination state in generations subsequent to the treated generation was independent of the presence of the transgenic allele (the recombination substrate under consideration) in the treated plant. We conclude that environmental factors lead to increased genomic flexibility even in successive, untreated generations, and may increase the potential for adaptation.

The Transcriptional Innate Immune Response to flg22. Interplay and Overlap with Avr Gene-Dependent Defense Responses and Bacterial Pathogenesis

Animals and plants carry recognition systems to sense bacterial flagellin. Flagellin perception in Arabidopsis involves FLS2, a Leu-rich-repeat receptor kinase. We surveyed the early transcriptional response of Arabidopsis cell cultures and seedlings within 60 min of treatment with flg22, a peptide corresponding to the most conserved domain of flagellin. Using Affymetrix microarrays, approximately 3.0% of 8,200 genes displayed transcript level changes in flg22 elicited suspension cultures and seedlings. FLARE (Flagellin Rapidly Elicited) genes mostly encode signaling components, such as transcription factors, protein kinases/phosphatases, and proteins that regulate protein turnover. Approximately 80% of flg22-induced genes were also up-regulated in Arabidopsis seedlings treated with cycloheximide. This suggests that many FLARE genes are negatively regulated by rapidly turned-over repressor proteins. Twenty-one tobacco Avr9/Cf-9 rapidly elicited (ACRE) cDNA full-length sequences were used to search for their Arabidopsis orthologs (AtACRE). We identified either single or multiple putative orthologs for 17 ACRE genes. For 13 of these ACRE genes, at least one Arabidopsis ortholog was induced in flg22-elicited Arabidopsis suspension cells and seedlings. This result revealed a substantial overlap between the Arabidopsis flg22 response and the tobacco Avr9 race-specific defense response. We also compared FLARE gene sets and genes induced in basal or gene-for-gene interactions upon different Pseudomonas syringae treatments, and infer that Pseudomonas syringae pv tomato represses the flagellin-initiated defense response.

Pattern-recognition receptors in plant innate immunity

Perception of pathogen-associated molecular patterns (PAMPs) constitutes the first layer of plant innate immunity and is referred to as PAMP-triggered immunity (PTI). For a long time, part of the plant community was sceptical about the importance of PAMP perception in plants. Genetic and biochemical studies have recently identified pattern-recognition receptors (PRRs) involved in the perception of bacteria, fungi and oomycetes. Interestingly, some of the structural domains present in PRRs are similar in plants and animals, suggesting convergent evolution. Lack of PAMP perception leads to enhanced disease susceptibility, demonstrating the importance of PAMP perception for immunity against pathogens in vivo. Recently, proteins with known roles in development have been shown to control immediate PRR-signalling, revealing unexpected complexity in plant signalling. Although many PAMPs recognised by plants have been described and more are likely to be discovered, the number of PRRs known currently is limited. The study of PTI is still in its infancy but constitutes a highly active and competitive field of research. New PRRs and regulators are likely to be soon identified.

The Arabidopsis Leucine-Rich Repeat Receptor–Like Kinases BAK1/SERK3 and BKK1/SERK4 Are Required for Innate Immunity to Hemibiotrophic and Biotrophic Pathogens

This work demonstrates that the leucine-rich receptor kinases (LRR-RKs) EFR and FLS2 form a ligand-induced complex with several LRR-RKs that belong to the SERK subfamily. Among these, BAK1 and BKK1 play an important role in responses to EF-Tu, flagellin, and other elicitors of plant defense and are required for full immunity to hemibiotrophic and biotrophic pathogens. Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) constitutes an important layer of innate immunity in plants. The leucine-rich repeat (LRR) receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN SENSING2 (FLS2) are the PRRs for the peptide PAMPs elf18 and flg22, which are derived from bacterial EF-Tu and flagellin, respectively. Using coimmunoprecipitation and mass spectrometry analyses, we demonstrated that EFR and FLS2 undergo ligand-induced heteromerization in planta with several LRR receptor-like kinases that belong to the SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, including BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1/SERK3 (BAK1/SERK3) and BAK1-LIKE1/SERK4 (BKK1/SERK4). Using a novel bak1 allele that does not exhibit pleiotropic defects in brassinosteroid and cell death responses, we determined that BAK1 and BKK1 cooperate genetically to achieve full signaling capability in response to elf18 and flg22 and to the damage-associated molecular pattern AtPep1. Furthermore, we demonstrated that BAK1 and BKK1 contribute to disease resistance against the hemibiotrophic bacterium Pseudomonas syringae and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis. Our work reveals that the establishment of PAMP-triggered immunity (PTI) relies on the rapid ligand-induced recruitment of multiple SERKs within PRR complexes and provides insight into the early PTI signaling events underlying this important layer of plant innate immunity.

Plant pattern recognition receptor complexes at the plasma membrane

A key feature of innate immunity is the ability to recognize and respond to potential pathogens in a highly sensitive and specific manner. In plants, the activation of pattern recognition receptors (PRRs) by pathogen-associated molecular patterns (PAMPs) elicits a defense programme known as PAMP-triggered immunity (PTI). Although only a handful of PAMP-PRR pairs have been defined, all known PRRs are modular transmembrane proteins containing ligand-binding ectodomains. It is becoming clear that PRRs do not act alone but rather function as part of multi-protein complexes at the plasma membrane. Recent studies describing the molecular interactions and protein modifications that occur between PRRs and their regulatory proteins have provided important mechanistic insight into how plants avoid infection and achieve immunity.

Plant pattern-recognition receptors

Plants are constantly exposed to would-be pathogens in their immediate environment. Yet, despite relying on innate immunity only, plants are resistant to most microbes. They employ pattern-recognition receptors (PRRs) for sensitive and rapid detection of the potential danger caused by microbes and pests. Plant PRRs are either surface-localized receptor kinases (RKs) or receptor-like proteins (RLPs) containing various ligand-binding ectodomains that perceive pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). In this review, I summarize our current knowledge of plant PRRs and their ligands, illustrating the multiple molecular strategies employed by plant PRRs to activate innate immune signaling to survive.