Flagellin from an incompatible strain of Pseudomonas avenae induces a resistance response in cultured rice cells

The host range of Pseudomonas avenae is wide among monocotyledonous plants, but individual strains can infect only one or a few host species. The resistance response of rice cells to pathogens has been previously shown to be induced by a rice-incompatible strain, N1141, but not by a rice-compatible strain, H8301. To clarify the molecular mechanism of the host specificity in P. avenae, a strain-specific antibody that was raised against N1141 cells and then absorbed with H8301 cells was prepared. When a cell extract of strain N1141 was separated by SDS-polyacrylamide gel electrophoresis and immunostained with the N1141 strain-specific antibody, only a flagellin protein was detected. Purified N1141 flagellin induced the hypersensitive cell death in cultured rice cells within 6 h of treatment, whereas the H8301 flagellin did not. The hypersensitive cell death could be blocked by pretreatment with anti-N1141 flagellin antibody. Furthermore, a flagellin-deficient N1141 strain lost not only the induction ability of hypersensitive cell death but also the expression ability of the EL2 gene, which is thought to be one of the defense-related genes. These results demonstrated that the resistance response in cultured rice cells is induced by the flagellin existing in the incompatible strain of P. avenae but not in the flagellin of the compatible strain.     

Flagellin from an incompatible strain of Acidovorax avenae mediates H2O2 generation accompanying hypersensitive cell death and expression of PAL,Cht-1, and PBZ1, but not of Lox in rice

Acidovorax avenae causes a brown stripe disease in monocot plants. We recently reported that a rice-incompatible strain of A. avenae caused hypersensitive cell death in rice and that the flagellin of the incompatible strain was involved in this response. The incompatible strain induced the rapid generation of H2O2 accompanying hypersensitive cell death and the expression of defense genes such as PAL, Cht-1, PBZ1, and LOX, whereas the compatible strain did not. The purified incompatible flagellin also induced the expression of PAL, Cht-1, and PBZ1, but LOX expression was not induced by the incompatible flagellin. PAL and LOX enzymatic activities were increased by inoculation with the incompatible strain, whereas only PAL activity was increased by the incompatible flagellin. Interestingly, the flagellin-deficient incompatible strain lost the ability to generate H2O2 and induce hypersensitive cell death, but PAL, Cht-1, and PBZ1 expression still were induced by inoculation with the deficient strain, suggesting that induction of these genes is regulated not only by flagellin but also by some other signal. Thus, the incompatible flagellin of A. avenae is a specific elicitor in rice, but it is not the only factor capable of inducing the rice defense system.     

Expression of a bacterial flagellin gene triggers plant immune responses and confers disease resistance in transgenic rice plants

Flagellin is a component of bacterial flagella and acts as a proteinaceous elicitor of defence responses in organisms. Flagellin from a phytopathogenic bacterium, Acidovorax avenae strain N1141, induces immune responses in suspension-cultured rice cells. To analyse the function of flagellin in rice, we fused the N1141 flagellin gene to the cauliflower mosaic virus 35S promoter and introduced it into rice. Many of the resulting transgenic rice plants accumulated flagellin at various levels. The transgenic rice developed pale spots in the leaves. The expression of a defence-related gene for phenylalanine ammonia-lyase was induced in the transgenic plants, and H(2)O(2) production and cell death were observed in some plants with high levels of gene expression, suggesting that the flagellin triggers immune responses in the transgenic rice. Transgenic plants inoculated with Magnaporthe grisea, the causal agent of rice blast, showed enhanced resistance to blast, suggesting that the flagellin production confers disease resistance in the transgenic rice.     

Glycosylation regulates specific induction of rice immune responses by Acidovorax avenae flagellin

Plants have a sensitive system that detects various pathogen-derived molecules to protect against infection. Flagellin, a main component of the bacterial flagellum, from the rice avirulent N1141 strain of the Gram-negative phytopathogenic bacterium Acidovorax avenae induces plant immune responses including H₂O₂ generation, whereas flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. To clarify the molecular mechanism that leads to these differing responses between the K1 and N1141 flagellins, recombinant K1 and N1141 flagellins were generated using an Escherichia coli expression system. When cultured rice cells were treated with recombinant K1 or N1141 flagellin, both flagellins equally induced H₂O₂ generation, suggesting that post-translational modifications of the flagellins are involved in the specific induction of immune responses. Mass spectrometry analyses using glycosyltransferase-deficient mutants showed that 1,600- and 2,150-Da glycans were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin induced immune responses in the same manner as N1141 flagellin. Site-directed mutagenesis revealed that glycans were attached to four amino acid residues (Ser¹⁷⁸, Ser¹⁸³, Ser²¹², and Thr³⁵¹) in K1 flagellin. Among mutant K1 flagellins in which each glycan-attached amino acid residue was changed to alanine, S178A and S183A, K1 flagellin induced a strong immune response in cultured rice cells, indicating that the glycans at Ser¹⁷⁸ and Ser¹⁸³ in K1 flagellin prevent epitope recognition in rice.     

The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception

Flagellin, the main building block of the bacterial flagellum, acts as a pathogen-associated molecular pattern triggering the innate immune response in animals and plants. In Arabidopsis thaliana, the Leu-rich repeat transmembrane receptor kinase FLAGELLIN SENSITIVE2 (FLS2) is essential for flagellin perception. Here, we demonstrate the specific interaction of the elicitor-active epitope flg22 with the FLS2 protein by chemical cross-linking and immunoprecipitation. The functionality of this receptor was further tested by heterologous expression of the Arabidopsis FLS2 gene in tomato (Lycopersicon esculentum) cells. The perception of flg22 in tomato differs characteristically from that in Arabidopsis. Expression of Arabidopsis FLS2 conferred an additional flg22-perception system on the cells of tomato, which showed all of the properties characteristic of the perception of this elicitor in Arabidopsis. In summary, these results show that FLS2 constitutes the pattern-recognition receptor that determines the specificity of flagellin perception.     

Biochemical and genetic requirements for function of the immune response regulator BOTRYTIS-INDUCED KINASE1 in plant growth, ethylene signaling, and PAMP-triggered immunity in Arabidopsis

Arabidopsis thaliana BOTRYTIS-INDUCED KINASE1 (BIK1) regulates immune responses to a distinct class of pathogens. Here, mechanisms underlying BIK1 function and its interactions with other immune response regulators were determined. We describe BIK1 function as a component of ethylene (ET) signaling and PAMP-triggered immunity (PTI) to fungal pathogens. BIK1 in vivo kinase activity increases in response to flagellin peptide (flg22) and the ET precursor 1-aminocyclopropane-1-carboxylic acid (ACC) but is blocked by inhibition of ET perception. BIK1 induction by flg22, ACC, and pathogens is strictly dependent on EIN3, and the bik1 mutation results in altered expression of ET-regulated genes. BIK1 site-directed mutants were used to determine residues essential for phosphorylation and biological functions in planta, including PTI, ET signaling, and plant growth. Genetic analysis revealed flg22-induced PTI to Botrytis cinerea requires BIK1, EIN2, and HUB1 but not genes involved in salicylate (SA) functions. BIK1-mediated PTI to Pseudomonas syringae is modulated by SA, ET, and jasmonate signaling. The coi1 mutation suppressed several bik1 phenotypes, suggesting that COI1 may act as a repressor of BIK1 function. Thus, common and distinct mechanisms underlying BIK1 function in mediating responses to distinct pathogens are uncovered. In sum, the critical role of BIK1 in plant immune responses hinges upon phosphorylation, its function in ET signaling, and complex interactions with other immune response regulators.     

Phosphoproteome analysis of Lotus japonicus roots reveals shared and distinct components of symbiosis and defense

Plant roots form an intracellular symbiosis with nitrogen-fixing bacteria while maintaining the capacity for defending themselves against bacterial pathogens. To investigate the molecular relationship between these opposing cellular responses, we compared changes in the root phosphoproteome of the legume Lotus japonicus occurring within minutes after perception of nodulation factor (NF), a symbiotic signaling molecule, to those elicited by flagellin peptide (flg22), a conserved pathogen-associated peptide motif present in flagellar protein of a wide range of bacteria. Phosphoproteins were visualized by autoradiography of two-dimensional polyacrylamide gels after in vivo labeling with 33P-orthophosphate. Comparisons of NF- and flg22-induced phosphoprotein patterns revealed signal-specific responses but also a surprisingly large overlap. Specificity of the responses was observed because the NF receptor kinases NFR1 and NFR5 were both required for NF- but not for flg22-mediated changes in the phosphoproteome. Moreover, NF did not stimulate an oxidative burst or activation of mitogen-activated protein kinases, two common markers for early defense responses that were induced by flg22. Inhibitor studies revealed that phosphorylation of at least some of the proteins in response to NF requires phospholipase D (PLD) whereas regulation of the flg22 phosphoproteome is PLD-independent. Although plant signal transduction during symbiosis and defense utilizes distinct components, phosphorylation of overlapping sets of proteins is achieved.     

How does SA signaling link the Flg22 responses?

Salicylic acid (SA) has a central role in activating plant resistance to pathogens. SA levels increase in plant tissue following pathogen infection and exogenous SA enhances resistance to a broad range of pathogens. To study the relevance of the SA signaling in the flg22 response, we investigated the responses of SA-related mutants to flg22, a 22-amino acid peptide of the flagellin bacterial protein. We identified SA as an important component of the flg22-triggered oxidative burst, a very early event after flg22 detection, and gene induction, an early event. SA acted partially by enhancing accumulation of FLS2 mRNA. We also provide new evidence that NPR1 play a role in SA-induced priming event that enhances the flg22-triggered oxidative burst, which is correlated with enhancement of the flg22-induced callose deposition. Based on these observations, we conclude that SA signaling is required for early as well as late flg22 responses.     

Molecular identification and characterization of the tomato flagellin receptor LeFLS2, an orthologue of <Emphasis Type="Italic">Arabidopsis</Emphasis> FLS2 exhibiting characteristically different perception specificities

Bacterial flagellin is known to stimulate host immune responses in mammals and plants. In Arabidopsis thaliana, the receptor kinase FLS2 mediates flagellin perception through physical interaction with a highly conserved epitope in the N-terminus of flagellin, represented by the peptide flg22 derived from Pseudomonas syringae. The peptide flg22 is highly active as an elicitor in many plant species. In contrast, a shortened version of the same epitope derived from Escherichia coli, flg15^{E coli}, is highly active as an elicitor in tomato but not in A. thaliana or Nicotiana benthamiana. Here, we make use of these species-specific differences in flagellin perception abilities to identify LeFLS2 as the flagellin receptor in tomato. LeFLS2 is most closely related to AtFLS2, indicating that it may represent the flagellin receptor of tomato. Expression of the LeFLS2 gene in Arabidopsis did not result in accumulation of its corresponding gene product, as indicated by experiments with LeFLS2-GFP fusions. In contrast, expression of LeFLS2-GFP fusions in N. benthamiana, a species that, like tomato, belongs to the Solanaceae, was obviously functional. N. benthamiana plants transiently expressing a LeFLS2-GFP fusion acquired responsiveness to flg15^{E coli} to which they are normally unresponsive. Thus, LeFLS2 encodes a functional, specific flagellin receptor, the first to be identified in a plant family other than the Brassicaceae. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The interplay between MAMP and SA signaling

There are two major modes for plant recognition of biotrophic microbial pathogens. In one mode, plant pattern recognition receptors (PRRs) recognize microbe associated molecular patterns (MAMPs, also called PAMPs), which are molecules such as flg22, a fragment of bacterial flagellin. In the other mode, the products of plant resistance (R) genes recognize pathogen effectors or host proteins modified by effectors. Salicylic acid (SA) -mediated defense responses are an important part of R gene-mediated resistance. It was not clear how these two signaling mechanisms interact with each other. Recently, we reported that treatment with flg22 triggered SA accumulation in Arabidopsis leaves. Disruptions of SA signaling components strongly affected MAMP-triggered gene expression responses. Flg22-triggered resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) was partly dependent on SA signaling. Our results demonstrated the importance of SA signaling in flg22-triggered resistance and, at the same time, the importance of some other signaling mechanism(s) in this resistance. Here we discuss potential signaling components of flg22-triggered SA accumulation and other signaling mechanisms potentially contributing to flg22-triggered resistance to Pst DC3000.Key words: arabidopsis, expression profiling, MAMP, PAD4, PAMP, salicylic acid (SA), SID2     

The Arabidopsis MAP kinase kinase MKK1 participates in defence responses to the bacterial elicitor flagellin

Plants sense pathogens through both pathogen-associated molecular patterns and recognition of race-specific virulence factors, which induce basal defence or an accelerated defence (often manifest in the form of local cell death), respectively. A mitogen-activated protein kinase (MAPK) module in Arabidopsis was previously proposed to signal from perception of the bacterial elicitor flagellin to the activation of basal defence-related genes. Here, we present evidence for a parallel MAPK-signalling pathway involved in the response to flg22, a peptide corresponding to the most conserved domain of flagellin. The endogenous Arabidopsis MAP kinase kinase MKK1 is activated in cells treated with flg22, phosphorylates the MAPK MPK4 in vitro, and activates it in vivo in protoplasts. In mkk1 mutant plants, the activation by flg22 of MPK4 and two other flg22-induced MAPKs (MPK3 and MPK6) is impaired. In the mkk1 mutant, a battery of both flg22-induced and flg22-repressed genes show altered expression, indicating that MKK1 negatively regulates the activity of flagellin-responsive genes. Intriguingly, in contrast to the mpk4 mutant, mkk1 shows no morphological anomalies and is compromised in resistance to both virulent and avirulent Pseudomonas syringae strains. Thus, the MKK1 signalling pathway modulates the expression of genes responding to elicitors and plays an important role in pathogen defence.     

Both the extracellular leucine-rich repeat domain and the kinase activity of FSL2 are required for flagellin binding and signaling in Arabidopsis

In Arabidopsis, activation of defense responses by flagellin is triggered by the specific recognition of the most conserved domain of flagellin, represented by the peptide flg22, in a process involving the FLS2 gene, which encodes a leucine-rich repeat serine/threonine protein kinase. We show here that the two fls2 mutant alleles, fls2-24 and fls2-17, which were shown previously to confer insensitivity to flg22, also cause impaired flagellin binding. These features are rescued when a functional FLS2 gene is expressed as a transgene in each of the fls2 mutant plants, indicating that FLS2 is necessary for flagellin binding. The point mutation of the fls2-17 allele lies in the kinase domain. A kinase carrying this missense mutation lacked autophosphorylation activity when expressed in Escherichia coli. This indicates that kinase activity is required for binding and probably affects the stability of the flagellin receptor complex. We further show that overexpression of the kinase-associated protein phosphatase (KAPP) in Arabidopsis results in plants that are insensitive to flagellin treatment, and we show reduced flg22 binding in these plants. Furthermore, using the yeast two-hybrid system, we show physical interaction of KAPP with the kinase domain of FLS2. These results suggest that KAPP functions as a negative regulator of the FLS2 signal transduction pathway and that the phosphorylation of FLS2 is necessary for proper binding and signaling of the flagellin receptor complex.     

Preliminary study on flg22-induced defense responses in female gametophytes of Saccharina japonica (Phaeophyta)

Flg22, which is the most conserved 22-amino acid peptide in the N-terminal part of flagellin, functions as an effective elicitor in higher plants. Marine algae and higher plants share some conserved characteristics on defense response pathways. Flg22-induced defense responses were investigated in female gametophytes of Saccharina japonica. Condensation of chloroplasts and thickened cell walls, as well as relatively stable structures of mitochondria and nucleus indicated that there were hypersensitive programmed cell death occurred after induction by flg22. By using luminol-dependent luminescence detection method, rapid release of H₂O₂ was detected in the induced female gametophytes and reached a peak concentration of about 46 μM at 2 h. Reactive oxygen species production was also observed histologically using the fluorescent dye 2′,7′-dichlorofluorescein diacetate, showing a consistent result with quantitative analysis of H₂O₂. Furthermore, results of antioxidant enzyme activities indicated that there was a trend in the order of catalase > superoxide dismutase > glutathione peroxidase. Finally, high level phenol content of cell-free extracts was found after flg22 induction. According to our results, flg22 could be an effective elicitor which could induce defense responses in female gametophytes of S. japonica.     

Biochemical and Morphological Features of Rice Cell Death Induced by Pseudomonas avenae

Pseudomonas avenae is a Gram-negative phytopathogenie bacteriumthat causes the symptom of a brown stripe in infected susceptibleplants. The host range of P. avenae is wide among the monocotyledonousplants, however, individual strains can infect only one or afew host species. A rice-incompatible strain, N1141, causedrapid cell death in sheath sections and in cultured rice cells.A rice-compatible strain, H8301, also induced cell death, however,this cell death in a compatible interaction was delayed comparedto the cell death induced by the N1141 incompatible strain.Inoculation of N1141 strain induced expression of EL2 gene whichis thought to be one of the defense-related gene. Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) of culturedrice cells showed that DNA cleavage occurred only in N1141-inoculatedrice cells. N1141 strain caused cytoplasmic condensation, shrinkage,and plasma membrane blebbing, all of which are important morphologicalcharacteristics of programmed cell death (PCD). In contrast,H8301 strain inoculated rice cells appeared to show weakeningof the cell wall instead of cytoplasm condensation, shrinkageand membrane blebbing. These results suggest that the rapidcell death of rice induced by the incompatible strain is characterizedas PCD. (Received May 22, 1999; Accepted July 22, 1999)     

DYNAMIN-RELATED PROTEIN DRP1A functions with DRP2B in plant growth,flg22-immune responses,and endocytosis

Ligand-induced endocytosis of the immune receptor FLAGELLIN SENSING2 (FLS2) is critical for maintaining its proper abundance in the plasma membrane (PM) to initiate and subsequently down regulate cellular immune responses to bacterial flagellin or flg22-peptide. The molecular components governing PM abundance of FLS2, however, remain mostly unknown. Here, we identified Arabidopsis (Arabidopsis thaliana) DYNAMIN-RELATED PROTEIN1A (DRP1A), a member of a plant-specific family of large dynamin GTPases, as a critical contributor to ligand-induced endocytosis of FLS2 and its physiological roles in flg22-signaling and immunity against Pseudomonas syringae pv. tomato DC3000 bacteria in leaves. Notably, drp1a single mutants displayed similar flg22-defects as those previously reported for mutants in another dynamin-related protein, DRP2B, that was previously shown to colocalize with DRP1A. Our study also uncovered synergistic roles of DRP1A and DRP2B in plant growth and development as drp1a drp2b double mutants exhibited severely stunted roots and cotyledons, as well as defective cell shape, cytokinesis, and seedling lethality. Furthermore, drp1a drp2b double mutants hyperaccumulated FLS2 in the PM prior to flg22-treatment and exhibited a block in ligand-induced endocytosis of FLS2, indicating combinatorial roles for DRP1A and DRP1B in governing PM abundance of FLS2. However, the increased steady-state PM accumulation of FLS2 in drp1a drp2b double mutants did not result in increased flg22 responses. We propose that DRP1A and DRP2B are important for the regulation of PM-associated levels of FLS2 necessary to attain signaling competency to initiate distinct flg22 responses, potentially through modulating the lipid environment in defined PM domains.

A plant-specific large dynamin GTPase is required for plant responses against bacterial pathogens and, with another dynamin, regulates the cell surface composition for plant growth and defense.     

Flagellin perception varies quantitatively in Arabidopsis thaliana and its relatives

Much is known about the evolution of plant immunity components directed against specific pathogen strains: They show pervasive functional variation and have the potential to coevolve with pathogen populations. However, plants are effectively protected against most microbes by generalist immunity components that detect conserved pathogen-associated molecular patterns (PAMPs) and control the onset of PAMP-triggered immunity. In Arabidopsis thaliana, the receptor kinase flagellin sensing 2 (FLS2) confers recognition of bacterial flagellin (flg22) and activates a manifold defense response. To decipher the evolution of this system, we performed functional assays across a large set of A. thaliana genotypes and Brassicaceae relatives. We reveal extensive variation in flg22 perception, most of which results from changes in protein abundance. The observed variation correlates with both the severity of elicited defense responses and bacterial proliferation. We analyzed nucleotide variation segregating at FLS2 in A. thaliana and detected a pattern of variation suggestive of the rapid fixation of a novel adaptive allele. However, our study also shows that evolution at the receptor locus alone does not explain the evolution of flagellin perception; instead, components common to pathways downstream of PAMP perception likely contribute to the observed quantitative variation. Within and among close relatives, PAMP perception evolves quantitatively, which contrasts with the changes in recognition typically associated with the evolution of R genes.