Activation Of Plant Defense Genes Research Articles

The Arabidopsis thaliana onset of leaf death 12 mutation in the lectin receptor kinase P2K2 results in an autoimmune phenotype

BackgroundPlant immunity relies on the perception of immunogenic signals by cell-surface and intracellular receptors and subsequent activation of defense responses like programmed cell death. Under certain circumstances, the fine-tuned innate immune system of plants results in the activation of autoimmune responses that cause constitutive defense responses and spontaneous cell death in the absence of pathogens.ResultsHere, we characterized the onset of leaf death 12 (old12) mutant that was identified in the Arabidopsis accession Landsberg erecta. The old12 mutant is characterized by a growth defect, spontaneous cell death, plant-defense gene activation, and early senescence. In addition, the old12 phenotype is temperature reversible, thereby exhibiting all characteristics of an autoimmune mutant. Mapping the mutated locus revealed that the old12 phenotype is caused by a mutation in the Lectin Receptor Kinase P2-TYPE PURINERGIC RECEPTOR 2 (P2K2) gene. Interestingly, the P2K2 allele from Landsberg erecta is conserved among Brassicaceae. P2K2 has been implicated in pathogen tolerance and sensing extracellular ATP. The constitutive activation of defense responses in old12 results in improved resistance against Pseudomonas syringae pv. tomato DC3000.ConclusionWe demonstrate that old12 is an auto-immune mutant and that allelic variation of P2K2 contributes to diversity in Arabidopsis immune responses.

Management of Black Root Disease-Causing Fungus Fusarium solani CRP1 by Endophytic Bacillus siamensis CNE6 through Its Metabolites and Activation of Plant Defense Genes.

Black root rot disease of Cicer arietinum L. is accountable for substantial loss in chickpea production worldwide. Endophytic Bacillus siamensis CNE6 has previously shown multifaceted plant growth-promoting, broad-spectrum antifungal, and chickpea plant-colonizing potential. In the present study, the strain Bacillus siamensis CNE6 was used for controlling black root rot disease caused by Fusarium solani CRP1 in chickpea. CNE6 showed strong antagonistic potential against CRP1 both in vivo and in vitro. Scanning electron microscopic studies indicated cellular deformation of CRP1 due to production of β-glucanase, protease, and other secondary metabolites. A total of five compounds were detected from the cell-free supernatant (CFS) of the ethyl acetate (EA) fraction of CNE6 through gas chromatography-mass spectrometry analysis. A confocal microscopic study demonstrated strong inhibition of biofilm formation of the pathogen CRP1 by the EA fraction of CFS of CNE6. Molecular docking analysis revealed that one compound, (2E)-6-methoxy-2-[(4-methoxyphenyl)methylidene]-2,3-dihydro-1-benzofuran-3-one, may inhibit the activity of lanosterol 14-alpha demethylase, which is involved in ergosterol biosynthesis and beta-tubulin assembling. In vivo experiments also showed the efficacy of CNE6 for increasing chickpea growth as well as upregulation of four defense genes (CHI1, PAMP, PR2B, and TF1082) upon pathogenic challenge. Thus, our results strongly suggest a positive role for CNE6 as a prospective biocontrol agent for combating Fusarium solani in chickpea. IMPORTANCE The present work was undertaken to explore an effective biocontrol agent against the destructive black root rot disease of chickpea. We have used an efficient bacterial endophyte, CNE6, which can colonize in the chickpea root system, produce secondary metabolites and enzymes to degrade pathogenic cellular integrity, inhibit pathogenic establishment by rupturing biofilm formation, and induce host immunity upon treatment. Interaction of the bacterial metabolite was also observed with lanosterol 14-alpha demethylase, which is an important component in fungal membrane functioning. Being an endophyte, Bacillus siamensis CNE6 fulfills a suitable criterion as a biocontrol agent to control black root rot disease in chickpea and has huge prospects for use commercially.

Inhibition of multiple defense responsive pathways by CaWRKY70 transcription factor promotes susceptibility in chickpea under Fusarium oxysporum stress condition

BackgroundSuppression and activation of plant defense genes is comprehensively regulated by WRKY family transcription factors. Chickpea, the non-model crop legume suffers from wilt caused by Fusarium oxysporum f. sp. ciceri Race1 (Foc1), defense response mechanisms of which are poorly understood. Here, we attempted to show interaction between WRKY70 and several downstream signaling components involved in susceptibility/resistance response in chickpea upon challenge with Foc1.ResultsIn the present study, we found Cicer arietinum L. WRKY70 (CaWRKY70) negatively governs multiple defense responsive pathways, including Systemic Acquired Resistance (SAR) activation in chickpea upon Foc1 infection. CaWRKY70 is found to be significantly accumulated at shoot tissues of susceptible (JG62) chickpea under Foc1 stress and salicylic acid (SA) application. CaWRKY70 overexpression promotes susceptibility in resistant chickpea (WR315) plants to Foc1 infection. Transgenic plants upon Foc1 inoculation demonstrated suppression of not only endogenous SA concentrations but expression of genes involved in SA signaling. CaWRKY70 overexpressing chickpea roots exhibited higher ion-leakage and Foc1 biomass accumulation compared to control transgenic (VC) plants. CaWRKY70 overexpression suppresses H2O2 production and resultant reactive oxygen species (ROS) induced cell death in Foc1 infected chickpea roots, stem and leaves. Being the nuclear targeted protein, CaWRKY70 suppresses CaMPK9-CaWRKY40 signaling in chickpea through its direct and indirect negative regulatory activities. Protein-protein interaction study revealed CaWRKY70 and CaRPP2-like CC-NB-ARC-LRR protein suppresses hyper-immune signaling in chickpea. Together, our study provides novel insights into mechanisms of suppression of the multiple defense signaling components in chickpea by CaWRKY70 under Foc1 stress.ConclusionCaWRKY70 mediated defense suppression unveils networking between several immune signaling events negatively affecting downstream resistance mechanisms in chickpea under Foc1 stress.

Identification and Sequence Comparison of Carbohydrate Oxidase Enzyme from Plant Sources

Plants produce hydrogen peroxide (H2O2) in response to wounding, pathogenic infection or other stresses, and it serves as a signal molecule that leads to the activation of plant defense genes. One enzyme capable of producing H2O2 in plants is carbohydrate oxidase. Previous work has provided information about highly homologous carbohydrate oxidase enzymes found in lettuce, sunflower and tobacco plants (Custers et al (2004) Plant J 39(2):147-160; Carter and Thornburg (2004) Plant Physiol. 134(1):460-469). This project looks to extend the knowledge base concerning the presence and function of plant carbohydrate oxidases. To begin, a number of plants from the greenhouse at The College of St. Scholastica as well as various food crops were tested for activity with a native in-gel assay. The in-gel assay probes for the presence of H2O2 using a horseradish peroxidase coupled reaction which produces a colored conjugate on any protein band that displays activity. Activity bands were obtained in-gel for plants including rabbit's foot fern, jade plant, tomato leaf and kale. Sequence data has been obtained by tandem mass spectrometry to confirm protein identity, and sequence comparison of enzymes from the literature and other databases will be presented. In future work, cloning target enzymes and overexpressing them in a recombinant system will provide a more robust way to access usable quantities of sample, allowing for kinetic characterization of individual enzymes. These studies will advance the understanding of plant defense mechanisms that utilize carbohydrate oxidase.

Chris Lamb: A Visionary Leader in Plant Science

Christopher John Lamb (1950-2009) made major contributions to the field of plant defense gene activation, particularly through his studies on signal transduction mechanisms. Between 1994 and 2004, he published a series of seminal papers that outlined the involvement of hydrogen peroxide, nitric oxide, lipid transfer proteins, and aspartic proteases as critical components of local and/or systemic resistance during plant-microbe interactions. Prior to this, he had been one of the first to establish the fact that induced defense responses resulted from transcriptional activation of sets of coordinately regulated genes. Chris obtained his B.S and PhD degrees in biochemistry from the University of Cambridge, United Kingdom, moving to the Botany School at the University of Oxford as a postdoctoral fellow in 1975 and to the Biochemistry Department in Oxford as a Departmental Demonstrator in 1978. He was appointed founding director of the Plant Biology Laboratory at the Salk Institute for Biological Studies in La Jolla, California in 1982, and occupied the last ten years of his life as Director of the John Innes Center, Norwich, United Kingdom. In spite of spending most of his career as a director at two of the world's most prestigious institutes, formal recognition of his achievements came late in life, with election to the Royal Society of London in 2008 and endowment of the honor of Commander of the British Empire (CBE) for his contributions to British plant science by Queen Elizabeth II in 2009. Sadly, Chris did not live to attend the official ceremony at which he would receive his CBE.

Overexpression of a wheat jasmonate-regulated lectin increases pathogen resistance

Jasmonates are known to induce the transcriptional activation of plant defense genes, which leads to the production of jasmonate-regulated proteins (JRP). We previously cloned and characterized a novel jacalin-like lectin gene ( Ta-JA1) from wheat ( Triticum aestivum L.), which codes a modular JRP with disease response and jacalin-related lectin (JRL) domains and is present only in the Gramineae family. The function of this protein is still unclear. Phylogenetic analysis indicated that Ta-JA1 and related proteins from cereals grouped together, which diverged from JRL with an additional N-terminal disease response domain. The recombinant Ta-JA1 proteins agglutinated rabbit erythrocytes, and this hemagglutination activity was preferentially inhibited by mannose. The Ta-JA1 protein was able to inhibit E. coli cell growth. Overexpression of Ta-JA1 in transgenic tobacco plants increased their resistance to infection by tobacco bacterial, fungal and viral pathogens. Our results suggest that Ta-JA1 belongs to a mannose-specific lectin, which may confer a basal but broad-spectrum resistance to plant pathogens. Ta-JA1 and its homologues in maize, rice, sorghum and creeping bentgrass may represent a new type of monocot lectin with a modular structure and diversity of physiological functions in biotic and abiotic stress responses.

Induction of Plant Defense Gene Expression by Plant Activators and Pseudomonas syringae pv. tomato in Greenhouse-Grown Tomatoes

Plant activators provide an appealing management option for bacterial diseases of greenhouse-grown tomatoes. Two types of plant activators, one that induces systemic acquired resistance (SAR) and a second that activates induced systemic resistance (ISR), were evaluated for control of Pseudomonas syringae pv. tomato and effect on plant defense gene activation. Benzothiadiazole (BTH, SAR-inducing compound) effectively reduced bacterial speck incidence and severity, both alone and in combination with the ISR-inducing product. Application of BTH also led to elevated activation of salicylic acid and ethylene-mediated responses, based on real-time polymerase chain reaction analysis of marker gene expression levels. In contrast, the ISR-inducing product (made up of plant growth-promoting rhizobacteria) inconsistently modified defense gene expression and did not provide disease control to the same level as did BTH. No antagonism was observed by combining the two activators as control of bacterial speck was similar to or better than BTH alone.

Characterization of a jasmonate-regulated wheat protein related to a beta-glucosidase-aggregating factor

Jasmonates are distributed throughout higher plants, where they play an important role in the activation of signal transduction pathways in response to wounding and pathogen attack. Jasmonates are known to induce the transcriptional activation of plant defense genes, produce jasmonate-regulated proteins (JRP). One class of 32 kDa JRP (designated as JRP-32 in this paper) is present in the Gramineae family, although the function of these proteins is still unclear. A cDNA was cloned from wheat ( Triticum aestivum L.) and designated as Ta-JA1. Sequence comparison indicated that Ta-JA1 encodes a JRP-32 protein. Ta-JA1 exists as a single-copy gene, but other genes with low sequence similarity to Ta-JA1 may be present in the wheat genome. The expression of Ta-JA1 was confined to stem tissues and barely detected in leaf and root tissues. Western blot analyses demonstrated that the recombinant Ta-JA1 protein cross-reacts with maize beta-glucosidase-aggregating factor (BGAF) antibody. Molecular modeling showed that Ta-JA1 and BGAF have a very similar three-dimensional structure. Protein structure analysis indicated that Ta-JA1 together with some related proteins (maize BGAF, wheat Ver2, WCI-1 and Hfr-1) contain two functional domains: a disease response domain and jacalin-related lectin (JRL) domain. A mannose-binding site was also well conserved in these proteins. The data support the hypothesis that JRP-32 and related proteins from Gramineae form a small protein family related to JRLs. This small protein family may have evolved from mannose-specific jacalin-related lectins (mJRLs) by developing a disease response domain in their N-terminus, which may have broadened the functional role of these proteins to include the plant defense response.

Activation of salicylic acid-induced protein kinase, a mitogen-activated protein kinase, induces multiple defense responses in tobacco.

The activation of mitogen-activated protein kinases (MAPKs) is one of the earliest responses in plants challenged by avirulent pathogens or cells treated with pathogen-derived elicitors. Expression of a constitutively active MAPK kinase, NtMEK2(DD), in tobacco induces the expression of defense genes and hypersensitive response-like cell death, which are preceded by the activation of two endogenous MAPKs, salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK). However, the roles that SIPK and WIPK each play in the process are unknown. Here we report that SIPK alone is sufficient to activate these defense responses. In tobacco leaves transiently transformed with SIPK under the control of a steroid-inducible promoter, the induction of SIPK expression after the application of dexamethasone, a steroid, leads to an increase of SIPK activity. The increase of SIPK activity is dependent on the phosphorylation of newly synthesized SIPK by its endogenous upstream kinase. In contrast, the expression of WIPK under the same conditions fails to increase its activity, even though the protein accumulates to a similar level. Studies using chimeras of SIPK and WIPK demonstrated that the C terminus of SIPK contains the molecular determinant for its activation, which is rather surprising because the N termini of SIPK and WIPK are more divergent. SIPK has been implicated previously in the regulation of both plant defense gene activation and hypersensitive response-like cell death based on evidence from pharmacological studies using kinase inhibitors. This gain-of-function study provided more direct evidence for its role in the signaling of multiple defense responses in tobacco.

A comparison of the effects of DNA-damaging agents and biotic elicitors on the induction of plant defense genes, nuclear distortion, and cell death.

Pea (Pisum sativum L. cv Alcan) endocarp tissue challenged with an incompatible fungal pathogen, Fusarium solani f. sp. phaseoli or fungal elicitors results in the induction of pathogenesis-related (PR) genes and the accumulation of pisatin, a phytoalexin. Essentially the same response occurs in pea tissue exposed to DNA-specific agents that crosslink or intercalate DNA. In this study, the effects of DNA-damaging agents were assessed relative to the inducible expression of several pea PR genes: phenylalanine ammonia lyase, chalcone synthase, and DRR206. Mitomycin C and actinomycin D mimicked the biotic elicitors in enhancing the expression of all three PR genes. The activities of these PR gene promoters, isolated from different plants, were evaluated heterologously in transgenic tobacco. It is remarkable that beta-glucuronidase expression was induced when plants containing the heterologous phenylalanine ammonia lyase, chalcone synthase, and DRR206 promoter-beta-glucuronidase chimeric reporter genes were treated by DNA-damaging agents. Finally, cytological analyses indicated that many of these agents caused nuclear distortion and collapse of the treated pea cells. Yet we observed that cell death is not necessary for the induction of the PR gene promoters assessed in this study. Based on these observations and previously published results, we propose that DNA damage or the associated alteration of chromatin can signal the transcriptional activation of plant defense genes.

Gentisic Acid As a Pathogen-Inducible Signal, Additional to Salicylic Acid for Activation of Plant Defenses in Tomato

Citrus exocortis viroid (CEVd) and tomato mosaic virus (ToMV), which produce a systemic non-necrotizing infection in tomato (Lycopersicon esculentum cv. Rutgers), strongly induced the accumulation of a phenolic compound that we have characterized as 2,5-dihydroxybenzoic acid (gentisic acid, GA) by nuclear magnetic resonance, following purification by high-performance liquid chromatography. Levels of free and total GA increased more than 150-fold in response to CEVd and ToMV infections. Unlike these non-necrotizing infections, the necrotizing reaction elicited by Pseudomonas syringae pv. syringae in this host did not produce any accumulation of GA. It is also shown that, in healthy leaf tissues, benzoic acid (BA) and salicylic acid (SA) were rapidly converted to GA, SA being the immediate precursor of GA, according to radiolabeling studies. Interestingly, exogenous GA elicited accumulation of the previously described CEVd-induced antifungal pathogenesis-related (PR) proteins P23, P32, and P34. These proteins were not induced by exogenous SA, which is able to elicit other CEVd-induced PR proteins in tomato. These results suggest that GA acts as a pathogeninduced signal, additional to SA, for activation of plant defense genes in tomato.

Molecular mechanisms of ethylene regulation of gene transcription

Ethylene‐regulated gene transcription has been studied in the context of plant defense, carnation flower petal senescence, and tomato fruit ripening. This review summarizes what is known about the molecular mechanisms of this process. Activation of plant defense genes in response to ethylene involves a promoter element called the GCC box, which interacts with ethylene‐responsive element‐binding proteins (EREBPs). Genes expressed in response to ethylene during petal senescence and fruit ripening use different promoter elements. For fruit ripening genes, two cooperative promoter elements are required for ethylene‐responsive transcription. DNA‐binding proteins of carnation and tomato that interact with ethylene‐responsive promoter elements have been studied, and in some cases cDNAs encoding such proteins have been isolated. The immediate goal of this work is to determine which DNA‐binding proteins are involved in mediating ethylene‐responsive gene transcription, and how they interact with other components of the ethylene signaling pathway. This work will lead to a more complete understanding of ethylene signal transduction.

The expression of the tobacco Tnt1 retrotransposon is linked to plant defense responses.

Activation of retrotransposons by stresses and external changes is common in all eukaryotic systems, including plants. The transcription of the tobacco Tnt1 retrotransposon was studied in its natural host as well as in Arabidopsis and tomato. It is activated by factors of microbial origin, by external stresses, and by viral, bacterial, and fungal attacks. Tnt1 expression is linked with the biological responses of the plant to the elicitor or to the pathogen attack and in particular with the early steps of the metabolic pathways leading to the activation of plant defense genes. In most cases, the basic features of Tnt1 regulation in tobacco are maintained in tomato and Arabidopsis, but some host-specific regulations were shown. The U3 region of the Tnt1 LTR contains the major cis-acting components of Tnt1 transcriptional activation in association with the plant defense responses. Furthermore, the Tnt1 U3 region, and especially the tandemly repeated BII boxes, contains several sequences similar to well-characterized motifs involved in the activation of several plant defense genes. The possible origin of Tnt1 regulatory sequences as well as the biological implications of Tnt1 activation by pathogen attacks are discussed.

Transcriptional activation of plant defense genes

Significant progress has been made in the characterization of disease resistance genes and receptors for pathogen avirulence signals and non-specific elicitors. Some components involved in elicitor-induced signal transduction have been identified. Phosphorylation of transcription factors has been found to be one of the mechanisms regulating their cellular localization, DNA binding and transcription activities for defense gene activation.

Purification and biochemical characterization of proteins which bind to the H-box cis-element implicated in transcriptional activation of plant defense genes

Purification and biochemical characterization of proteins which bind to the H-box cis-element implicated in transcriptional activation of plant defense genes

Purification and biochemical characterization of proteins which bind to the H‐box cis‐elementimplicated in transcriptional activation of plant defense genes

The H-box (CCTACC(N)7CT(N)4A), which occurs three times within the -154 to -42 region of the bean chalcone synthase chs15 promoter, is important for developmental regulation of chs15, and induction of chs15 and coordinately regulated defense genes by elicitors and other stress stimuli. Two protein factors, KAP-1 and KAP-2, which recognize conserved features in the H-box motif, were purified from bean cell suspension cultures by a combination of ion exchange chromatography and DNA affinity chromatography. KAP-1 is a 97 kDa polypeptide, whereas KAP-2 comprises two polypeptides of 76 and 56 kDa. KAP-1 and KAP-2 also differ in the sensitivity of their DNA-bound forms to trypsin. Dephosphorylation of KAP-1 or KAP-2 affects the mobility of the protein/H-box binding complex in gel shift assays but does not inhibit DNA binding. Elicitation of bean cell suspensions with glutathione does not affect the total cellular activities of KAP-1 or KAP-2, but causes a rapid increase in the specific activities of both factors in the nuclear fraction, consistent with a role for these factors in the signal pathway for elicitor induction of chs15 and related defense genes.

Elicitor Recognition and Signal Transduction in Plant Defense Gene Activation

Plants defend themselves against pathogen attack by activating a whole set of defense responses, most of them relying on transcriptional activation of plant defense genes. The same responses are induced by treatment of plant cells with elicitors released from the pathogen or from the plant surface. Several plant/elicitor combinations have been used successfully as experimental systems to investigate the molecular basis of plant defense responses. Receptor-like structures on the plasma membrane of plant cells appear to bind the elicitors. Thereby, intracellular signal transduction chains are initiated which finally result in the activation of plant defense genes. A better understanding of the molecular mechanisms of early processes in plant defense responses, as provided by these studies, may in the long term help to develop environmentally safe plant protection methods for agriculture.

Transcriptional activation of plant defense genes by fungal elicitor, wounding, and infection.

Activation of plant defense genes was investigated by analysis of transcripts completed in vitro by isolated nuclei. Elicitor treatment of suspension-cultured bean (Phaseolus vulgaris L.) cells caused marked transient stimulation of transcription of genes encoding apoproteins of cell wall hydroxyproline-rich glycoproteins (HRGP) and the phenylpropanoid biosynthetic enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), concomitant with the onset of rapid accumulation of the respective mRNAs and hence expression of the phytoalexin (PAL, CHS), lignin (PAL), and HRGP defense responses. While there was a lag of 2 h prior to stimulation of HRGP gene transcription, induction of the transcription of PAL and CHS genes occurred within 5 min of elicitor treatment. Induction of transcription of PAL, CHS, and HRGP genes was also observed in wounded hypocotyls and in infected hypocotyls during race-cultivar-specific interactions with the fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. Transcriptional activation occurred not only in directly infected tissue but also in distant, hitherto uninfected tissue, indicating intercellular transmission of an endogenous signal for defense gene activation. It is concluded that transcriptional activation of defense genes characteristically underlies induction of the corresponding defense responses and expression of disease resistance.

Transcriptional activation of plant defense genes by fungal elicitor, wounding, and infection.

Activation of plant defense genes was investigated by analysis of transcripts completed in vitro by isolated nuclei. Elicitor treatment of suspension-cultured bean (Phaseolus vulgaris L.) cells caused marked transient stimulation of transcription of genes encoding apoproteins of cell wall hydroxyproline-rich glycoproteins (HRGP) and the phenylpropanoid biosynthetic enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), concomitant with the onset of rapid accumulation of the respective mRNAs and hence expression of the phytoalexin (PAL, CHS), lignin (PAL), and HRGP defense responses. While there was a lag of 2 h prior to stimulation of HRGP gene transcription, induction of the transcription of PAL and CHS genes occurred within 5 min of elicitor treatment. Induction of transcription of PAL, CHS, and HRGP genes was also observed in wounded hypocotyls and in infected hypocotyls during race-cultivar-specific interactions with the fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. Transcriptional activation occurred not only in directly infected tissue but also in distant, hitherto uninfected tissue, indicating intercellular transmission of an endogenous signal for defense gene activation. It is concluded that transcriptional activation of defense genes characteristically underlies induction of the corresponding defense responses and expression of disease resistance.