Signal perception and transduction in plant defense responses.
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Animals↗
Biomedical subjects
D F Klessig
Publications and source records attributed to D F Klessig.
Identification of an Arabidopsis locus required for resistance to turnip crinkle virus.
Inoculation of turnip crinkle virus (TCV) into a (TCV)-resistant line of Arabidopsis thaliana, Di-17, results in the development of a hypersensitive response (HR) on the inoculated leaves. In contrast, an HR does not occur when leaves of the TCV-susceptible Di-3 line or the susceptible ecotypes Columbia (Col-0), or Landsberg erecta (Ler) are inoculated. Genetic analysis of progeny from crosses between Di-17 and either Di-3, Col-0 or Ler demonstrates that the development of an HR is regulated by a single dominant nuclear locus, herein designated HRT. Using progeny from a Di-17 x Col-0 cross, HRT was mapped to chromosome 5, where it is tightly linked to the DFR locus. We also demonstrate that a variety of resistance-associated phenomena, including the TCV-induced accumulation of salicylic acid, camalexin and autofluorescent cell-wall material, correlate with the HR, suggesting the possibility that HRT is required for their activation.
Arabidopsis↗
Development of necrosis and activation of disease resistance in transgenic tobacco plants with severely reduced catalase levels.
Numerous studies argue that salicylic acid (SA) is an important component of the plant signal transduction pathway(s) leading to disease resistance. The discovery that the SA-binding protein is a catalase, whose activity is blocked by SA, led to the proposal that one of SA's modes of action is to inhibit this H2O2-degrading enzyme and thus elevate H2O2 levels. To test this model, an attempt was made to mimic the action of SA by reducing the synthesis of catalase using antisense RNA technology. Analyses of transgenic tobacco plants that expressed the tobacco catalase 1 (cat1) or catalase 2 (cat2) gene in an antisense orientation indicate that there is no correlation between modest to high levels of reduction in catalase activity and activation of plant defenses such as pathogenesis-related (PR)-1 protein synthesis. However, three independent antisense catalase transgenic plants (ASCAT1 Nos 16, 17, and 28), which exhibited the most severe reduction in catalase activity (approximately 90% or more), developed chlorosis or necrosis on some of their lower leaves. These same leaves accumulated very high levels of PR-1 proteins and showed enhanced resistance to tobacco mosaic virus. Necrosis and elevated SA, which appear to result from severe depression of catalase levels, may be responsible for the induction of these defense responses.
Amino Acid Sequence↗
Characterization of a salicylic acid-insensitive mutant (sai1) of Arabidopsis thaliana, identified in a selective screen utilizing the SA-inducible expression of the tms2 gene.
Salicylic acid (SA) plays an important signaling role in the resistance of many plants to pathogen invasion. Increases in endogenous SA levels have been associated with the hypersensitive response as well as systemic acquired resistance (SAR). SA also induces the expression of a subset of the pathogenesis-related (PR) genes. However, relatively little is known about the events occurring subsequent to SA accumulation during a resistance response. In order to identify mutations in components of the SA signal transduction pathway, we have developed a genetic screen in Arabidopsis thaliana that utilizes the Agrobacterium tumefaciens tms2 gene as a counter-selectable marker. SA-inducible expression of the tms2 gene from the tobacco PR-1a promoter confers sensitivity to alpha-naphthalene acetamide (alpha-NAM), resulting in inhibition of root growth in germinating transgenic Arabidopsis seedlings. Mutants in which root growth is insensitive to alpha-NAM have been selected from this PR-1a:tms2 transgenic line with the expectation that a subset will lack a regulatory component downstream of SA. The sail mutant so identified expressed neither the PR-1a:tms2 transgene nor the endogenous Arabidopsis PR-1, PR-2, and PR-5 genes in response to SA. These genes also were not induced in sai1 by 2,6-dichloroisonicotinic acid (INA) or benzothiadiazole (BTH), two chemical inducers of SAR. As expected of a mutation acting downstream of SA, sai1 plants accumulate SA and its glucoside in response to infection with an avirulent pathogen and are more susceptible to this avirulent pathogen than the wild-type parent. sai1 is allelic to npr1, a previously identified SA-noninducible mutation. The recessive nature of the noninducible sai1 mutation suggests that the wild-type SAI1 gene acts as a positive regulator in the SA signal transduction pathway.
Amidohydrolases↗
Salicylic acid activates a 48-kD MAP kinase in tobacco.
The involvement of phosphorylation/dephosphorylation in the salicylic acid (SA) signal transduction pathway leading to pathogenesis-related gene induction has previously been demonstrated using kinase and phosphatase inhibitors. Here, we show that in tobacco suspension cells, SA induced a rapid and transient activation of a 48-kD kinase that uses myelin basic protein as a substrate. This kinase is called the p48 SIP kinase (for SA-Induced Protein kinase). Biologically active analogs of SA, which induce pathogenesis-related genes and enhanced resistance, also activated this kinase, whereas inactive analogs did not. Phosphorylation of a tyrosine residue(s) in the SIP kinase was associated with its activation. The SIP kinase was purified to homogeneity from SA-treated tobacco suspension culture cells. The purified SIP kinase is strongly phosphorylated on a tyrosine residue(s), and treatment with either protein tyrosine or serine/threonine phosphatases abolished its activity. Using primers corresponding to the sequences of internal tryptic peptides, we cloned the SIP kinase gene. Analysis of the SIP kinase sequence indicates that it belongs to the MAP kinase family and that it is distinct from the other plant MAP kinases previously implicated in stress responses, suggesting that different members of the MAP kinase family are activated by different stresses.
Amino Acid Sequence↗
The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance.
The cpr5 mutant was identified from a screen for constitutive expression of systemic acquired resistance (SAR). This single recessive mutation also leads to spontaneous expression of chlorotic lesions and reduced trichome development. The cpr5 plants were found to be constitutively resistant to two virulent pathogens, Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2; to have endogenous expression of the pathogenesis-related gene 1 (PR-1); and to have an elevated level of salicylic acid (SA). Lines homozygous for cpr5 and either the SA-degrading bacterial gene nahG or the SA-insensitive mutation npr1 do not express PR-1 or exhibit resistance to P. s. maculicola ES4326. Therefore, we conclude that cpr5 acts upstream of SA in inducing SAR. However, the cpr5 npr1 plants retained heightened resistance to P. parasitica Noco2 and elevated expression of the defensin gene PDF1.2, implying that NPR1-independent resistance signaling also occurs. We conclude that the cpr5 mutation leads to constitutive expression of both an NPR1-dependent and an NPR1-independent SAR pathway. Identification of this mutation indicates that these pathways are connected in early signal transduction steps and that they have overlapping functions in providing resistance.
Arabidopsis↗
Isolation and characterization of a tobacco mosaic virus-inducible myb oncogene homolog from tobacco.
Salicylic acid (SA) plays an important role in signaling the activation of plant defense responses against pathogen attack including induction of pathogenesis-related (PR) proteins. To gain further insight into the SA-mediated signal transduction pathway, we have isolated and characterized a tobacco mosaic virus (TMV)-inducible myb oncogene homolog (myb1) from tobacco. The myb1 gene was induced upon TMV infection during both the hypersensitive response and development of systemic acquired resistance in the resistant tobacco cultivar following the rise of endogenous SA, but was not activated in the susceptible cultivar that fails to accumulate SA. The myb1 gene was also induced by incompatible bacterial pathogen Pseudomonas syringae pv. syringae during the hypersensitive response. Exogenous SA treatment rapidly (within 15 min) activated the expression of myb1 in both resistant and susceptible tobacco cultivars with the subsequent induction of PR genes occurring several hours later. Biologically active analogs of SA and 2,6-dichloroisonicotinic acid (a synthetic functional analog of SA), which induce PR genes and enhanced resistance, also activated the myb1 gene. In contrast, biologically inactive analogs were poor inducers of myb1 gene expression. Furthermore, the recombinant Myb1 protein was shown to specifically bind to a Myb-binding consensus sequence found in the promoter of the PR-1a gene. Taken together, these results suggest that the tobacco myb1 gene encodes a signaling component down-stream of SA that may participate in transcriptional activation of PR genes and plant disease resistance.
Amino Acid Sequence↗
Salicylic acid is a modulator of tobacco and mammalian catalases.
Salicylic acid (SA) plays a key role in the establishment of resistance to microbial pathogens in many plants. The discovery that SA inhibits catalase from tobacco led us to suggest that H2O2 acts as second messenger to activate plant defenses. Detailed analyses of SA's interaction with tobacco and mammalian catalases indicate that SA acts as an electron donor for the peroxidative cycle of catalase. When H2O2 fluxes were relatively low (1 microM/min or less), SA inhibited catalase, consistent with its suggested signaling function via H2O2. However, significant inhibition was only observed at 100 microM SA or more, a level reached in infected, but not in uninfected, leaves. This inhibition was probably due to siphoning catalase into the slow peroxidative reaction. Surprisingly, SA was also able to protect catalase from inactivation by damaging levels of H2O2 (lower millimolar range), which is generally assumed to reflect accumulation of inactive ferro-oxy intermediates. SA did so by supporting or substituting for the protective function of catalase-bound NADPH. These results add new features to SA's interaction with heme enzymes and its in vivo redox properties. Thus, SA, in addition to its proposed signaling function, may also have an important antioxidant role in containing oxidative processes associated with plant defense responses.
Animals↗
Identification of a salicylic acid-responsive element in the promoter of the tobacco pathogenesis-related beta-1,3-glucanase gene, PR-2d.
The tobacco pathogenesis-related PR-2d gene encodes an acidic beta-1,3-glucanase. Expression of the PR-2d: uidA(GUS) chimeric gene is induced in leaves undergoing the hypersensitive resistance response to tobacco mosaic virus and after treatment with salicylic acid (SA), a chemical believed to play an important role(s) in disease resistance. We have constructed transgenic tobacco plants which carry various segments of the PR-2d promoter fused to a heterologous core 35S promoter driving the uidA(GUS) reporter gene. Their analysis indicates that sequences from -364 to -288 upstream of the PR-2d transcription start site confer a high level of activation by SA (20-fold). Mutations within this sequence, located between -339 and -333, depressed SA activation. This region is also required for the SA-inducibility of a truncated PR-2d:GUS chimeric gene. Contained within this region is a 25 bp element located between -348 and -324 which was specifically recognized by nuclear factors from tobacco leaves. No conclusive differences were observed in the ability of proteins in nuclear extracts from water-treated versus SA-treated plants to bind to this cis element in vitro.
Binding Sites↗
Inhibition of ascorbate peroxidase by salicylic acid and 2,6-dichloroisonicotinic acid, two inducers of plant defense responses.
In recent years, it has become apparent that salicylic acid (SA) plays an important role in plant defense responses to pathogen attack. Previous studies have suggested that one of SA's mechanisms of action is the inhibition of catalase, resulting in elevated levels of H2O2, which activate defense-related genes. Here we demonstrate that SA also inhibits ascorbate peroxoidase (APX), the other key enzyme for scavenging H2O2. The synthetic inducer of defense responses, 2,6-dichloroisonicotinic acid (INA), was also found to be an effective inhibitor of APX. In the presence of 750 microM ascorbic acid (AsA), substrate-dependent IC50 values of 78 microM and 95 microM were obtained for SA and INA, respectively. Furthermore, the ability of SA analogues to block APX activity correlated with their ability to induce defense-related genes in tobacco and enhance resistance to tobacco mosaic virus. Inhibition of APX by SA appears to be reversible, thus differing from the time-dependent, irreversible inactivation by suicide substrates such as p-aminophenol. In contrast to APX, the guaiacol-utilizing peroxidases, which participate in the synthesis and crosslinking of cell wall components as part of the defense response, are not inhibited by SA or INA. The inhibition of both catalase and APX, but not guaiacol peroxidases, supports the hypothesis that SA-induced defense responses are mediated, in part, through elevated H2O2 levels or coupled perturbations of the cellular redox state.
Ascorbate Peroxidases↗
Two inducers of plant defense responses, 2,6-dichloroisonicotinec acid and salicylic acid, inhibit catalase activity in tobacco.
2,6-Dichloroisonicotinic acid (INA) and salicylic acid (SA) are potent inducers of plant defense responses including the synthesis of pathogenesis-related (PR) proteins and the development of enhanced disease resistance. A soluble SA-binding protein has been purified from tobacco with an affinity and specificity of binding that suggest it is a SA receptor. Recently, this protein has been shown to be a catalase whose enzymatic activity is inhibited by SA binding. We have proposed that the resulting increase in intracellular levels of reactive oxygen species plays a role in the induction of defense responses such as PR protein gene expression. Here we report that INA, like SA, binds the SA-binding protein/catalase and inhibits its enzymatic activity. In fact, the dose-response curves for inhibition of catalase by these two compounds are similar. Furthermore, the ability of both INA analogues and SA derivatives to bind and inhibit tobacco catalase correlates with their biological activity to induce PR-1 gene expression and enhance resistance to tobacco mosaic virus. Comparison of the structures of INA, SA, and their analogues reveals several common features that appear to be important for biological activity. Thus, these results not only suggest that INA and SA share the same mechanism of action that involves binding and inhibition of catalase but also further indicate an important role for reactive oxygen species in the induction of certain plant defense responses. This is supported by the demonstration that INA-mediated PR-1 gene activation is suppressed by antioxidants.
Journal Article↗
Identification of a high-molecular-weight cellular protein complex containing the adenovirus DNA binding protein.
The adenovirus DNA binding protein (DBP) functions in a number of diverse processes that include viral DNA replication and host range determination. A subpopulation of DBP has been found to exist in a high-molecular-weight (HMWDBP) complex of greater than 650 kDa. This complex was stable to high salt and low levels of detergent in the presence of DTT at 4 degrees. While the complex prepared in high salt was devoid of nucleic acid, it retained its ability to bind to ssDNA. Electron microscopic (EM) analysis in the presence of M13 ssDNA verified its interaction with ssDNA. The complex exists as particles approximately 25-30 nm based on EM analysis of negatively stained samples in the absence of DNA. No viral replication components were detected in the HMWDBP complex, suggesting that it is not a viral replication complex. The HMWDBP complex prepared under a variety of infection conditions contained a consistent set of proteins. HMWDBP complex was also detected in the absence of viral infection in gmDBP6 cells (a HeLa cell derivative that expresses DBP from the dexamethasone-inducible MMTV promoter). These results suggest that the components of the complex, other than DBP, are host encoded.
Adenoviruses, Human↗
Programmed cell death in plants: a pathogen-triggered response activated coordinately with multiple defense functions.
In plants, the hypersensitive response (HR) to pathogens involves rapid cell death, which is hypothesized to arise from the activation of a cell death program. We describe mutant A. thaliana plants that contain lesions in a single accelerated cell death (ACD) gene called ACD2 and that bypass the need for pathogen exposure to induce the HR. acd2 plants that develop spontaneous lesions show typical HR characteristics both within the necrotic tissue and within the healthy part of the plant, including: modification of plant cell walls, resistance to bacterial pathogens, and accumulation of defense-related gene transcripts, the signal molecule salicylic acid and an antimicrobial compound. We propose that the ACD2 gene is involved in a pathway(s) that negatively regulates a genetically programmed HR.
Apoptosis↗
The salicylic acid signal in plants.
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Biological Transport↗
Salicylic acid, active oxygen species and systemic acquired resistance in plants.
Infection of plants, particularly by a necrotizing pathogen, usually induces a long-lasting, broad-based, systemic resistance to secondary pathogen attack. Many studies implicate salicylic acid as an essential signal in the development of such systemic acquired resistance in several plant species. Salicylic acid appears to mediate plant defence by binding to and inhibiting catalase, thus increasing the concentration of H(2)O(2) and other active oxygen species. Active oxygen species may then act as second messengers that induce plant defence gene expression, analogous to their activation of gene expression in mammalian cells.
Journal Article↗
A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance.
Systemic acquired resistance (SAR) is a nonspecific defense response in plants that is associated with an increase in the endogenous level of salicylic acid (SA) and elevated expression of pathogenesis-related (PR) genes. To identify mutants involved in the regulation of PR genes and the onset of SAR, we transformed Arabidopsis with a reporter gene containing the promoter of a beta-1,3-glucanase-encoding PR gene (BGL2) and the coding region of beta-glucuronidase (GUS). The resulting transgenic line (BGL2-GUS) was mutagenized, and the M2 progeny were scored for constitutive GUS activity. We report the characterization of one mutant, cpr1 (constitutive expressor of PR genes), that was identified in this screen and shown by RNA gel blot analysis also to have elevated expression of the endogenous PR genes BGL2, PR-1, and PR-5. Genetic analyses indicated that the phenotype conferred by cpr1 is caused by a single, recessive nuclear mutation and is suppressed in plants producing a bacterial salicylate hydroxylase, which inactivates SA. Furthermore, biochemical analysis showed that the endogenous level of SA is elevated in the mutant. Finally, the cpr1 plants were found to be resistant to the fungal pathogen Peronospora parasitica NOCO2 and the bacterial pathogen Pseudomonas syringae pv maculicola ES4326, which are virulent in wild-type BGL2-GUS plants. Because the cpr1 mutation is recessive and associated with an elevated endogenous level of SA, we propose that the CPR1 gene product acts upstream of SA as a negative regulator of SAR.
Arabidopsis↗
Induction, modification, and perception of the salicylic acid signal in plant defence.
Endogenous salicylic acid (SA) levels increase and several families of pathogenesis-related genes (including PR-1 and PR-2) are induced during the resistance response of tobacco to tobacco mosaic virus (TMV) infection. We have found that at a temperature (32 degrees C) that prevents the induction of PR genes and resistance, the increases in SA levels were eliminated. However, when the resistance response was restored by shifting inoculated plants to lower temperatures, SA levels increased dramatically and preceded PR-1 gene expression and necrotic lesion formation associated with resistance. SA was also found in a conjugated form whose levels increased in parallel with the free SA levels. This SA beta-glucoside (SAG) was as active as SA in inducing PR-1 gene expression. PR-1 gene induction by SAG was preceded by a transient release of SA. The existence of a mechanism that releases SA from SAG suggests a possible role for SAG in the maintenance of systemic acquired resistance. Previously, we identified a soluble salicylic acid-binding protein (SABP) in tobacco whose properties suggest that it may play a role in transmitting the SA signal during plant defence responses. This SABP has been purified 250-fold by sequential chromatography on DEAE-Sephacel, Sephacryl S-300, Blue Dextran-Agarose and Superose 6. Several monoclonal antibodies (mAbs) raised against the highly purified SABP immunoprecipitated the SA-binding activity and a 280 kDa protein. This 280 kDa protein also co-purified with the SA-binding activity during the various chromatography steps, suggesting that it was responsible for binding SA. Immunoblot analysis with the SABP-specific mAbs also detected the 280 kDa protein in highly purified preparations of SABP. However, in crude homogenates these mAbs only recognized a 57 kDa protein. These and other results suggest that SABP is a multimeric complex which contains, at least, a 57 kDa protein and whose components are readily cross-linked during purification.
Carrier Proteins↗
Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid.
A complementary DNA encoding a salicylic acid (SA)-binding protein has been cloned. Its properties suggest involvement in SA-mediated induction of systemic acquired resistance (SAR) in plants. The sequence of the protein is similar to that of catalases and the protein exhibits catalase activity. Salicylic acid specifically inhibited the catalase activity in vitro and induced an increase in H2O2 concentrations in vivo. H2O2 or compounds, such as SA, that inhibit catalases or enhance the generation of H2O2, induced expression of defense-related genes associated with SAR. Thus, the action of SA in SAR is likely mediated by elevated amounts of H2O2.
Amino Acid Sequence↗