Browsing by Person "Pfitzner, Artur J. P."

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Analyse von Pathogenresistenzmechanismen in Tomate (Solanum lycopersicum L.)
(2008) Gerhardts, Anja; Pfitzner, Artur J. P.
For many organisms plants serve as a source of nutrients and energy, but because of their static location they are exposed to various harmful environmental influences. Due to this factor they have developed complex defence mechanisms e. g. for protection against pathogens. An important aspect of these defence mechanisms is the expression of intrinsic resistance genes (R) that detect pathogenic avirulence gene products (Avr) thereby causing a hypersensitive response (HR) in the infected cells and consequently inhibiting the systemic infection of the plant. In this work the resistance genes Tm-2 and Tm-2² of tomato were isolated, cloned and sequenced. The allelic R genes are members of the CC-NBS-LRR group of resistance genes, which is widely spread in plants, and differ only in four amino acids. This is surprising because using resistance breaking ToMV strains Weber et al. (2004) showed that both resistance gene products interact differently with the movement protein (30 kDa MP = Avr) of the virus. To gain further insight into this phenomenon of different pathogen detection, chimeric exchange constructs (A1 and A2) were designed through restriction in the region between the NBS and the LRR domain. These four constructs were used for transformation of MM tomatoes as well as NN and nn tobacco plants. The expression of the resistance gene constructs in MM an nn lines did not confer the expected resistance to ToMV. Nevertheless in older infected nn transformants a formation of spontaneous necrosis was observed, which indicates a delayed development of HR. One possible explanation could be that the presence of only the resistance gene product is not sufficient to detect the viral movement protein and that other host cellular components are involved in this process (as in the guard hypothesis by Dangl and Jones, 2001). This assumption is supported by our yeast two hybrid interaction experiments which showed that a direct interaction of Tm-2 and 30 kDa MP can be excluded. For the NN transformants differences in functionality of the constructs was observed. While NN/Tm-2 and NN/A2 plants showed extreme resistance to ToMV wild type (ToMV0) and the Tm-2² resistance breaking strain ToMV2², the Tm-2² and A1 constructs conferred less resistance to ToMV0 and the Tm-2 resistance breaking strain ToMV1-2. This finding also supports the assumption that there is a difference in pathogen detection between the two alleles. Furthermore it shows that the detection takes place within the LRR region because the exchange construct that behaves in the same way as the endogenous resistance gene carries the C-terminal LRR domain of this allele. The hydroxycinnamoyl-CoA:tyramine N-(Hydroxycinnamoyl)transferase (THT) was found to be another candidate for transmission of pathogen resistance during HR (Gerhardts, 2003). Our in vivo results show that the products of the THT enzymatic reaction induced during HR does not only have an antimicrobiotic effect on the pathogen (von Roepenack-Lahaye et al., 2003; Newman et al., 2001) but also has an apoptotic effect on the plant cell itself.
Analysis of the structure of tomato mosaic virus movement protein based on virus host interactions
(2011) Tanwir, Fariha; Pfitzner, Artur J. P.
Viruses are obligatory plant pathogens causing sever diseases, and ultimately great losses in crop yield. Plant viruses, once entered in the cell, make use of host machinery for its own replication and moves from one cell to the other. Natural resistance against virus attack is achieved by the presence of resistance genes (R genes). R genes recognize viral avirulence (Avr) factors in elicitor-receptor manner to initiate resistance cascade. In tomato, the resistance genes Tm-I, Tm-2 and Tm-22 are used to protect the plants against infection by tomato mosaic virus.Tm-2 and Tm-22 require recognition of the viral 30kDa movement protein (MP) for triggering resistance response. Sequence analysis of Tm-2 and Tm-22 resistance breaking viruses have shown an amino-acid exchange at position 133 (E>K) is found in all Tm-2 resistance breaking virus strains, whereas, amino-acid exchange at position 130 (K>E) is associated with Tm-22 resistance breaking phenotype (Calder and Palukaitis, 1992). This suggests a physical interaction between resistance genes and 30kDa MP. In the present study, a unique Split GFP approach is used to analyse the structure and localization of different domains of 30kDa MP in S. cerviceae and N. benthamiana. Different deletion mutants were fused between two non-overlapping halves of GFP and expressed. Results showed that both N and C terminus as well as the middle part of 30kDa MP (aa 80-150) is present in the cytoplasm with two integral membrane loops. These findings are in contrast with previous in-vitro results, which suggest that middle part of 30kDa MP is present in ER lumen, whereas N and C terminus in cytoplasm (Brill et al., 2000). Fluorescence microscopy revealed that GFP fused 30kDa MP deletion mutants were localized on the cytoplasmic side of plasmamembrane and near plasmodesmata. Membrane association of fusion protein confirmed the proper folding and functionality of deletion mutants. Therefore, the structural model of ToMV 30kDa MP has to be revised. Secondly, to identify the host factors involved in resistance mechanism, initiated by Tm-2 and Tm-22 resistance genes, tomato mosaic virus based vectors were constructed. Two different types of in-vivo transcription vectors were constructed, one containing both right and left border of the T-DNA (pBinSLN) and one without the right border (pBinSLN-RB). Self replication of these vectors were analysed in N. benthamiana, N. tabacum and S. lycopersicum. It was found that the deletion of RB does not affect virus replication, when agro-infiltrated in N. benthamiana. pBinSLN-RB was used further for the isolation of a stable and vigorous Tm-2 and Tm-22 resistance breaking ToMV strain through a novel selection scheme. ToMV2-22 contains two amino-acid exchanges at position 54(N>D) and 133(E>K). ToMV2-22 is the first mutant strain of ToMV, which can escape both Tm-2 and Tm-22 resistance simultaneously.
Die Bedeutung von AQUAPORIN INTERACTOR 1 (AQI1) für die Zelltodregulation in Pflanzen
(2014) Glink, Eva Katharina; Pfitzner, Artur J. P.
Programmed cell death (PCD) is an important process during development, senescence and pathogen defence in plants and in animals. It is a genetically regulated and targeted cell suicide of single cells, for benefit of the whole organism. In plants, PCD is of great importance, especially in the course of the “hypersensitive response” (HR). For protecting themselves against harmful intruders, infected plant cells are directly deposed of by PCD. The developing local lesions act as a barrier between host plant and pathogen. This prevents the systemic expansion of biotrophic pathogens within the whole plant. The induction of PCD involves complex signal transduction pathways. Reactive oxygen species (ROS), in particular H₂O₂, play an important role as signal molecules during PCD. The transport of H₂O₂ across cell membranes is conducted by aquaporins. As the vitality of cells depends on intracellular H₂O₂-levels, a spatiotemporal control of this H₂O₂-transport is indispensable. AQUAPORIN INTERACTOR 1 (AQI1) was isolated as a potential regulator of the channel function of aquaporins. AQI1 is a plant protein with sequence homology to the mammal aminoacylase 1. It is known, that aminoacylases catalyse the hydrolysis of acyl-amino acids. However, the physiological function of these enzymes is still unclear. This study represents the first characterisation of an aminoacylase (AQI1) in plants. The physiological function of AQI1 as a regulator of aquaporins, as well as the underlying molecular mechanisms, have been analysed. In addition to deacetylation of amino acids, a second function of the protein AQI1 was discovered. AQI1 interferes with the channel activity of aquaporins by protein-protein interaction. In this way, AQI1 is able to inhibit the H₂O₂-, and to a certain extent also the H₂O-influx, through aquaporins. Probably, this happens by blocking the aquaporinpore. Due to this function, AQI1 is a major component in cell death regulation in plants. During the „hypersensitive response“ (HR), which is induced as a result of pathogen attack, AQI1 accumulates to high levels to prevent the influx of toxic amounts of H₂O₂ into neighbouring cells. This ensures a local control of PCD. In addition, AQI1 seems to be involved in regulation of senescence processes. It could be demonstrated, that AQI1 accumulates in a gradient from juvenile to senescent leaves, due to degradation in older tissues. By this age-dependent accumulation, AQI1 could contribute to the vitality of leaves, by preventing the influx of excessive amounts of H₂O₂ into the cell.
Die Bedeutung von Aquaporin interagierenden Proteinen für die Zelltodregulation bei Pflanzen und Tieren
(2020) Straub, Anna Katharina; Pfitzner, Artur J. P.
Hydrogen peroxide plays a crucial role as a signalling molecule in the induction of cell death in plants and animals. To mediate signalling and induce apoptosis in a cell, hydrogen peroxide molecules need to be transported across different membranes to their target site. In plants and animals, integral membrane proteins called aquaporins, facilitate the transport of hydrogen peroxide between cell compartments by channelling the signalling molecule across membranes. Plant aquaporins are regulated by proteins called Aquaporin Interactor 1 and 2 (AQI1 and AQI2). AQI2 is a plant homolog of AQI1. Both proteins function as inhibitors of aquaporins by binding to the channels resulting in prevention of water and hydrogen peroxide influx. Aquaporin Interactor 1 binds preferentially to the aquaporin tonoplast intrinsic protein TIP1.1, while Aquaporin Interactor 2 exhibits a binding preference to the aquaporin plasma membrane intrinsic protein PIP2.2. Aquaporin Interactor 1 is located in the vacuole or associated to the tonoplast membrane. In contrast, results obtained for Aquaporin Interactor 2 suggest that it is located in the apoplast. This is compatible with the hypothesis that tonoplast aquaporins can be regulated by AQI1, whereas plasma membrane aquaporins on the other hand are regulated by AQI2. The enzyme Aminoacylase 1 is known to hydrolyse N-acetylated amino acids. It is a zinc-binding metalloenzyme with a wide range of substrates. However, its preferred substrate is N-acetyl-Methionine. N-acetyl-Methionine can also be hydrolysed by the plant homolog AQI1. The plant enzyme also needs metal ions as co-factors. Of note, no aminoacylase activity was found for AQI2. Experiments using aqi1 knock-out mutants of Arabidopsis thaliana and Nicotiana tabacum clearly show, that hydrolysis of N-acetyl-Methionine can only be accomplished by AQI1. However, the aminoacylase activity of AQI1 is not needed for the ability to bind to aquaporins. The data show that the aminoacylase activity and the ability to bind aquaporins are two separate functions of the protein Aquaporin Interactor 1. Based on current knowledge, it must be assumed, that AQI2 acts only as an aquaporin-regulating protein. After pathogen attack an increased aminoacylase activity could be detected in the affected plant tissue. This AQI1 induction can be observed both after agrobacteria infiltration and after infection with the tobacco mosaic virus. This suggests a role for AQI1 in pathogen defence. Another aquaporin interacting protein is BHRF1, an anti-apoptotic protein originating from the Epstein-Barr virus. To date, an interaction between BHRF1 and aquaporins could only be detected with plant aquaporins. Transgenic BHRF1 N. tabacum plants show spontaneously occurring cell death events apparent by necrotic plant tissue. These necrotic areas are caused by BHRF1 interacting with plant aquaporins and several proteins of the G-protein signalling pathway inducing cell death. By binding to the aquaporins, BHRF1 is able to replace the endogenous aquaporin interaction partners AQI1 and AQI2. Thus, a precise aquaporin regulation by endogenous AQI1 and AQI2 is no longer guaranteed. Moreover, results show that BHRF1 can bind the Arabidopsis glucose sensor AtRGS1 (regulator of G-protein signalling). AtRGS1 is a combination of a G-protein coupled receptor and a RGS protein. The RGS domain causes the hydrolysis of GTP bound to the Gα subunit. Further experiments showed, an interaction of BHRF1 with human RGS proteins. Therefore, BHRF1 could also have a possible effect on G-protein signalling in humans. The results of this study demonstrate the importance of a precise regulation of aquaporins in cell death regulation. Deregulation caused by viral BHRF1, leads to cell death events. BHRF1 presumably competes with the endogenous interaction partners of aquaporins and of the G-protein-signalling pathway, ultimately resulting in the deregulation of various signalling pathways.
Die Bedeutung von Aquaporinen und ihren Interaktionspartnern für die Zelltodregulation in Pflanzen
(2011) Hoch, Tanja; Pfitzner, Artur J. P.
Programmed cell death (PCD, apoptosis) is an induced cell suicide process that plays an important role during the differentiation and pathogen defense responses of plants and animals. BHRF1 (?BamHI fragment H rightward open reading frame no. 1?) is a cell-death modulating protein of the Epstein-Barr virus (EBV), a human lymphotrophic herpes virus. The expression of BHRF1 in transgenic plants led to the formation of necrotic lesions. Further experiments showed that BHRF1 associated necrotic lesions are caused due to stress, senescence and pathogen defense responses. Yeast-two-hybrid-screening of a tobacco cDNA library identified two different aquaporins as partners for interacting with BHRF1. Aquaporins were identified as water channels/carriers within red blood cells, but are also present in all other organisms. Over the last years, more information was gathered indicating that, apart from transporting water, aquaporins had other functional activities. E. g. Henzler and Steudle (2000) demonstrated that aquaporins can act as hydrogen peroxide channels in the algae Chara corallina. Furthermore, publications by Bienert et al. (2006), indicating that aquaporins in plants as well as in animals are also able to transport H2O2. Hydrogen peroxide and other reactive oxygen species (ROS) have long been recognized as important signal molecules during the pathogen defense response in plants, therefore establishing a logical connection between cell death and aquaporins for the first time. It was assumed that the aquaporins NtPIP2.2a, NtPIP2.2b und NtTIP1.1a identified during the yeast-two-hybrid-screen can act as H2O2 channels. In further experiments it could indeed be established that these aquaporins have the ability to transport H2O2 in yeast cells. Yeasts expressing aquaporins could be influenced in their H2O2 sensitivity by the expression of BHRF1. BHRF1 without transmembrane domain (BHRF1deltaTMwt) led to an enhanced H2O2 sensitivity and also to an increase in cell death. In addition, the transient expression of aquaporin could induce necrotic lesions and cell death in Nicotiana benthamiana. Deletion experiments identified a common binding domain for interacting with BHRF1 in these aquaporins. This binding domain consists of the conserved region containing the first NPA motive (?loop? B) that is also half of one pore. Further studies showed that BHRF1 interacts with all kinds of different aquaporins from plants, animals (rAQP8) and humans (hAQP1). BHRF1 most likely binds with the alpha3 helix to the highly conserved NPA region of aquaporins. A cellular protein showing sequence homology to M20 proteases and aminoacylases was isolated when looking for interaction partners of aquaporins in plants. Like BHRF1, this protein binds to the conserved NPA region of the aquaporins. Although the cellular substrate for this protein has to be found yet, an interesting observation was made. Co-expression of the isolated aminoacylase with NtTIP1.1a or NtPIP2.2b in Nicotiana benthamiana led to the inhibition of cell death induced by these aquaporins.
Charakterisierung der akzessorischen Proteine vom felinen Coronavirus (FCoV) mit monoklonalen Antikörpern
(2014) Lemmermeyer, Tanja; Pfitzner, Artur J. P.
Little is known about the expression and function of the FCoV accessory proteins 3a, 3b, 3c, 7a and 7b. These proteins of FIPV strain 79-1146 were investigated in the present study. The obtained results are outlined: 1. The accessory FCoV proteins 3a, 3c and 7b were expressed in E. coli with a C-terminal his-tag. Furthermore the proteins 3a, 3b, 3c, the C-terminal half part of 3c, 7a, 7b as well as 7b without the amino acids 1 to 17 (7bdeltaSS) were expressed as fusion proteins with an N-terminal GST-tag and a C-terminal his-tag. The purification of all fusion proteins was performed by Ni2+ ion affinity chromatography under denaturing conditions. 2. To generate monoclonal antibodies the purified fusion proteins 3c and 7b were used for immunization of mice. ELISA screenings were established which enabled the identification of hybridoma cells that produce mabs against 3c and 7b. 3. The characterization of the anti-3c mabs led to the identification of regions in the C-terminus of the protein. The 3c protein could not be detected in an eukaryotic inducible expression system (Tet-on cell line BHKFIPV-3c) and also not in FCoV-infected cells. The anti-7b mabs bound within the region of amino acids 58 to 75 and reacted with a recombinant 7b fusion protein of a serotype I FCoV. 4. The expression of the 7b protein in infected cells was confirmed by western blot. An N-glycosylation site is located within the binding region. After incubation with tunicamycin the signal obtained with the anti-7b mabs was considerably stronger. Again after tunicamycin treatment the 7b protein was detected in the cytoplasm of infected cells by indirect immunofluorescence. The 7b protein colocalized partially with the ER. 5. The recombinant 7b protein was detected with all of the anti-FIPV 79-1146 sera and the ascites, but not with the anti-FECV 79-1683 sera. In contrast the recombinant fusion proteins 3a, 3b, 3c and 7a were not detected with the analyzed anti-FCoV sera.
Effects of overexpression of NIMIN genes on salicylic acid-mediated PR-1 gene activation and phenotype in Nicotiana benthamiana (Domin)
(2013) Masroor, Ashir; Pfitzner, Artur J. P.
Systemic acquired resistance (SAR) is a whole plant resistance mechanism, launched after initial exposure to a necrotizing pathogen. At molecular level, SAR is characterized by elevated level of plant hormone salicylic acid (SA) and induction of pathogenesis-related (PR) proteins. During SAR, SA signal is transduced through regulatory protein NPR1 (Non-Expressor of PR Genes1; also known as NIM1 or SAI1) leading to the induction of the SAR marker PR-1. Present data strongly suggest that the SA signal is directly perceived by NPR1. NPR1 interacts with two classes of proteins. DNA binding TGA factors link the SA sensor NPR1 to the as-1 like cis-acting elements present in the promoter region of PR-1 gene. In addition, NPR1 interacts with NIM1-interacting (NIMIN) proteins. In Arabidopsis, there are four NIMIN genes, i.e., NIMIN1, NIMIN1b, NIMIN2 and NIMIN3. Initially, it was hypothesized that, although structurally related to each other, NIMIN proteins might play diverse functions during SAR response. Indeed, it has been shown that the NIMIN genes are expressed differentially and that the encoded proteins interact differentially with NPR1. Based on these observations, NIMIN proteins have gained much attention. The functional significance of NIMIN proteins in SAR pathway has been addressed in overexpression studies. Overexpression of NIMIN1 yielded strong suppression of PR gene induction and enhanced susceptibility to a bacterial pathogen in transgenic Arabidopsis. Apart from NIMIN1, the functional significance of other Arabidopsis NIMIN family members has not yet been addressed. Therefore, present research is conducted to explore the biological significance of other NIMIN family members from Arabidopsis as well as tobacco. To this end, transient gene expression in a N. benthamiana reporter line containing a -1533PR-1apro:GUS construct was employed. The research achievements of this work are listed below. 1. The N. benthamiana infiltration procedure was optimized for reliable determination of -1533PR-1apro:GUS reporter gene activation in presence of different Agrobacterium strains. 2. After optimization, transient gene expression system (TGES) was successfully used to uncover the functional significance of NIMIN proteins on SA-mediated PR-1a gene induction. NIMIN1 and NIMIN1b are categorized as strong, NIMIN3 as an intermediate and NIMIN2 as a non-suppressor of SA-mediated PR-1a reporter gene activation. 3. Interestingly, suppressing NIMIN1, NIMIN1b and NIMIN3 all contain an EDF (glutamic acid, aspartatic acid, phenylalanine) motif. Therefore, EDF mutants were generated in NIMIN1 and NIMIN3, i.e., NIMIN1 E94A D95V and NIMIN3 E63A D64V, respectively. Yeast two-hybrid (Y2H) data show that NIMIN1 E94A D95V still interacts with NPR1, while NIMIN3 E63A D64V interaction with NPR1 could not be validated due to undetectable accumulation of the mutant fusion protein in yeast. In the TGES, NIMIN1 E94A D95V and NIMIN3 E63A D64V were not able to suppress the SA-mediated PR-1a promoter activation. The data support the fact that the EDF motif may have a function in NIMIN proteins interaction with NPR1, thereby, regulating PR-1 gene induction. 4. EAR domain is generally considered as a repression domain and also exists in NIMIN proteins. The deletion mutants, i.e., NIMIN1 1/137 and NIMIN1b 1/135 still suppress the SA-induced -1533PR-1apro:GUS gene activation. On the other hand, NIMIN1 L138A L140A and NIMIN3 L108A L110A do not suppress the SA-mediated reporter gene induction. But that is because of low overall accumulation of mutant proteins in N. benthamiana leaf tissues. Thus, the data support the view that EAR domain is not the only repressional domain active in NIMIN proteins. 5. Like Arabidopsis, tobacco also contains NIMIN genes. During this study, a novel NIMIN gene from tobacco, NIMIN-like1, was cloned and characterized. Using Y2H analyses, it was shown that NIMIN-like1 binds to NgNPR1 and that interaction is sensitive to SA. Thus, NIMIN-like1 falls into the tobacco NIMIN family. Thereafter, functional significance of diverse tobacco NIMIN proteins for their effects on SA-mediated PR-1a gene induction via TGES was carried out. NIMIN2c and NIMIN-like1 are categorized as strong suppressors, whereas NIMIN2a is a weak suppressor of SA-mediated PR-1a reporter gene induction. 6. NIMIN1 and NIMIN3 overexpression manifests cell death in N. benthamiana, and cell death is accompanied by the accumulation of H2O2. No correlation was found between NIMIN proteins binding intensity to NPR1 and cell death. Similarly, no correlation was found between PR-1a reporter gene suppression and cell death. The data support the view that the EDF and EAR motifs are not involved in cell death phenomenon. Based on previous and data gathered in this study, a model for the hypothetical play of sequential interaction of NIMIN proteins with NPR1 in course of SAR is presented.
Expression and functional domains of Arabidopsis and tobacco NIM1-INTERACTING (NIMIN) genes
(2021) Saur, Mathias; Pfitzner, Artur J. P.
Systemic acquired resistance (SAR) is an important defense mechanism in plants initiated after exposure to biotrophic pathogens. SAR is characterized by accumulation of PR proteins in non-infected tissues, as well as increased concentrations of the phytohormone salicylic acid (SA). SA is directly perceived by NPR1, the key regulator of SAR. Through interaction with TGA transcription factors and NIM1-INTERACTING (NIMIN) proteins, NPR1 mediates the SA-dependent induction of PR1 gene expression. The Arabidopsis genome contains four NIMIN genes – NIMIN1 (N1), N1b, N2, and N3 – but members of the NIMIN family can also be found in other higher plants. While NIMIN proteins share their general domain architecture and a C-terminal EAR motif, they differ in other aspects. NIMIN genes are expressed differentially during pathogen infection and development. NIMIN proteins can be subdivided based on their NPR1-interaction motifs, the DXFFK and the EDF motif. N1-type proteins harbor both domains, while N2-type and N3-type proteins carry only the DXFFK or the EDF motif, respectively. Accordingly, NIMIN proteins interact differentially with NPR1: N1, N1b and N2 bind to the C-terminal moiety while N3 binds to the N-terminus. Overexpression studies revealed a role for the N1 and N3 proteins in the transcriptional repression of PR1 gene induction. Strikingly, infiltrated plants overexpressing Arabidopsis N1 and N3 or tobacco N2c also manifest significantly accelerated cell death. These numerous differences indicate diverse functions of NIMIN proteins during SAR establishment and beyond. The objective of this work was to further characterize differences between NIMIN proteins from Arabidopsis and tobacco regarding biochemical properties and biological functions with special emphasis on their cell death promoting activity. For this purpose, reporter constructs harboring promoter and coding regions from Arabidopsis and tobacco NIMIN genes were analyzed in transient gene expression experiments in Nicotiana benthamiana and in transgenic tobacco plants. Functional domains were examined using the introduction of targeted mutations to study their significance for NIMIN protein function. The following results were obtained: 1. The N1b 1135 promoter region is functional and two reporter genes under its control, GUS and the proapoptotic Bax, are active during transient overexpression. In transgenic tobacco plants the N1b promoter is not responsive to chemical induction by SA or its functional analog BTH and phenotypical studies showed no expression during plant development. To what extent the N1b gene is expressed in plants must therefore remain open. 2. Transient overexpression of Arabidopsis N1 and N3 and tobacco N2 type genes N2c and N2-like (FS) results in accelerated cell death. This enhanced emergence of cell death is associated with strong protein accumulation. In transgenic tobacco plants overexpression of the N1, N2c and FS genes is also accompanied by emergence of cell death, especially in the flower area, and low seed production. The affected plants often display defects in growth and leaf morphology. 3. The ability to promote cell death requires the C-terminal EAR motif, a transcriptional repression domain. Mutation of the EAR motif in N1, N2c and FS significantly reduces the emergence of cell death. In yeast the EAR motifs of N1 and N3 interact with a N-terminal fragment of the transcriptional co-repressor TOPLESS (TPL). Transient overexpression of this TPL1/333 fragment also induces cell death but coexpression with N1 or N3 reduces cell death emergence, indicating that NIMIN proteins not only affect NPR1 but also modulate the activity of TPL. 4. The enhanced emergence of cell death mediated by overexpression of NIMIN genes and Bax interferes with measurement of SA induced activity of the PR1 promoter. However, using EAR motif mutans with reduced cell death emergence, like the N1 F49/50S E94A D95V EAR mutant, which is also unable to bind NPR1, allows the analysis of the transcriptional repression of the PR1 promoter mediated by cell-death promoting NIMIN proteins. 5. N1 contains a conserved N-terminal domain (N1nT) of 15 amino acids which regulates its accumulation. In N-terminal position, this domain functions autonomously with other NIMIN proteins and Venus, increasing their accumulation. Mutational analysis has not yet revealed reliance on certain sequences. Presence of the N-terminal methionine is not required for function of the N1nT domain hinting at a function at the mRNA level. NIMIN proteins are multifunctional and could perform different functions through their conserved domains. The results indicate that NIMIN proteins, through their interaction with TOPLESS, could also affect other hormone-dependent signal pathways. While the exact mechanism remains unclear, the enhanced protein accumulation bestowed by the N1nT domain of N1 could allow for more effective study of poorly accumulating proteins.
Funktionelle Bedeutung unterschiedlicher NPR-Proteine für die Salicylsäure-abhängige Genexpression im Rahmen der systemisch aktivierten Resistenz in Arabidopsis thaliana und Nicotiana tabacum
(2018) Konopka, Evelyn Maria Anna Hedwig; Pfitzner, Artur J. P.
Systemic acquired resistance (SAR) is an important mechanism for plants to protect themselves against biotrophic pathogens. The main characteristic of SAR is the accumulation of PR proteins in non-infected, distal leaf tissues. The expression of PR1 genes in tobacco (Nt) and Arabidopsis (At) is induced by salicylic acid (SA). NPR1 is the central regulatory protein of SAR. In cooperation with NIMIN proteins and TGA transcription factors, NPR1 controls the induction of PR1 gene expression dependent on SA. NIMIN proteins function as negative regulators of PR1 gene expression, whereas TGA transcription factors mediate binding to SA responsive cis-acting as1-like elements of PR1 promotors. The perception of SA occurs at the C-terminus of NPR1, where SA sensitive binding of NIMIN1 (N1) and NIMIN2 (N2) proteins takes place at the N1/2 binding domain. The arginine within the conserved LENRV motif is significantly involved in the perception of SA. Mutation of the arginine leads to loss of SA sensitivity of NIMIN binding to AtNPR1 and NtNPR1. Arabidopsis possesses three other NPR protein family members with a similar domain structure as NPR1: AtNPR2, AtNPR3 and AtNPR4. In tobacco, only NtNPR1 and NtNPR3 exist. SA dependent reactions for AtNPR3, AtNPR4 and NtNPR3 in yeast are also known. While the NIMIN2 binding to the NtNPR3 C-terminus is negatively affected by SA, the C-termini of AtNPR3 and AtNPR4 respond to SA with a structural rearrangement. The C-terminal domains LENRV-like domain and N1/2 binding domain exhibit a SA inducible affinity to each other. The aim of this work was to obtain new insights of the function of NPR1 and its homologues and paralogues in Arabidopsis and tobacco regarding the SA dependent gene expression, the mechanism of SA perception and signal transduction. The heterologous yeast system was applied for the analysis of the biochemical properties of At and Nt NPR proteins. The relevance of Nt NPR proteins in planta was analyzed by using CRISPR/Cas9 generated mutants. The following results were obtained: 1. A SA dependent reaction for AtNPR2 could be shown. The spontaneous interaction with TGA transcription factors of clades II and III is reinforced in a SA dependent manner. 2. The arginine within the LENRV-like motifs of At and Nt NPR proteins is substantially involved in SA perception. Mutation of the arginine leads to a total loss of the SA dependent reactions of At and Nt NPR proteins, without changing further biochemical capabilities. 3. SA dependent reactions of the analyzed At and Nt NPR proteins are also inducible by structural analogues of SA. Especially dichlorinated compounds like 3,5 dichloroanthranilic acid (3,5 DCA) and 3,5 dichlorobenzoic acid proved to be very potent, but also BTH (benzothiadiazole) and INA (2,6-dichloroisonicotinic acid), known inducers of PR1 gene expression, show direct effects on NPR proteins. AtNPR4 is an exception. It is the only member of the protein family that is specific for SA. IC50 and EC50 values indicate that NtNPR proteins are more sensitive to SA than AtNPR proteins and that 3,5 DCA is more effective than SA. 4. AtNPR2, AtNPR3 and AtNPR4 can interact with TGA transcription factors of clades II and III as strongly as AtNPR1. However, the paralogues are not able to interact with members of the NIMIN protein family in spite of a conserved N1/2 binding domain in the C-termini. Binding to NIMIN proteins and forming ternary complexes with NIMIN proteins and TGA transcription factors are unique to AtNPR1. 5. Chimeric interactions show that the LENRV domain of NtNPR3 is pivotal for the C-terminus to rearrange in response to SA as found previously for AtNPR1. Thus, the comparison of other known biochemical characteristics suggests a higher functional similarity of AtNPR1 to NtNPR3 than to NtNPR1. 6. Analysis of NtNPR1 and NtNPR3 mutants generated by CRISPR/Cas9 shows that deletions and insertions were introduced at specific positions within the first exon of the target genes, which result in breakdown of translation or transition of the open reading frame. Although PR1 gene expression is dependent on NtNPR1, reduction of accumulation of PR1 proteins was not observed after induction in plants with mutated NtNPR1 gene. In contrast, individuals with NtNPR3 as target gene for mutagenesis show a significant reduction of PR1 accumulation in independent lines of the F2 generation after induction. These results are in accordance with the biochemical analysis. At and Nt NPR1 proteins and other NPR family members are sensitive to SA. The arginine within the LENRV-like domain mediates the perception of SA. In fact, only AtNPR1 and not its paralogues functions as a positive regulator of SAR. In tobacco, NPR3 is probably the functional homologue of AtNPR1, even though NtNPR1 exhibits a higher sequence similarity to AtNPR1.
Regulation von NIMIN- und PR1-Genen aus Arabidopsis thaliana (L.) Heynh. und Nicotiana tabacum (L.) in der Salicylat-abhängigen Pathogenabwehr
(2009) Hermann, Meike; Pfitzner, Artur J. P.
Systemic acquired resistance (SAR) is an important defense mechanism of plants against a broad range of pathogens. NPR1 acts as a central regulator controlling the salicylic acid (SA)-dependent formation of SAR through interaction with TGA transcription factors leading to the induction of ?pathogenesis-related? (PR) proteins. The SA-activated expression of the PR1 genes in Arabidopsis thaliana (At) and Nicotiana tabacum (Nt) depends on cis-acting as-1-like elements with a TGACG sequence. This dissertation studies the functional relevance of NIMIN proteins and SA-dependent PR gene induction using the analysis of gene regulation. Arabidopsis has four NIMIN-genes ? N1, N2, N3 and N1b which interact independently of TGA transcription factors with NPR1. N1 and N2 have a common interaction motif and bind to the C-terminus of AtNPR1, whereas N3 binds to the N-terminus of AtNPR1. The binding site for the TGA transcription factors is located relatively central in the AtNPR1 protein. The analysis of the NIMIN gene expression in the SA-dependent signaling pathway of SAR as well as their possible involvement in the Jasmonic (JA) signaling network ought to offer new aspects for understanding the regulation of plant pathogen defense. The relevance of different as-1-like elements was studied by establishing a yeast one-hybrid system. N1b is likely to be an inactive pseudogene. Neither could transcripts be detected in untreated, SA- or JA-treated Arabidopsis plants nor was the construct N1b[GUS] with the 1135 bp 5?-region able to induce reporter gene expression in transgenic tobacco plants. Expression of N3 occurs constitutively at low levels and independently of NPR1. Treatment with SA or JA does not lead to induction of N3. Likewise, the N3 promoter is not affected by treatment with SA, JA, TMV and phytohormones. Reporter gene expression of the N3 promoter occurs constitutively in transgenic tobacco seedlings. In contrast, N1 and N2 are clearly SA-induced. After SA induction, the expression of N2 is immediate and long-lasting and regulated independently of NPR1. N1 is expressed transiently at a later point in time and is NPR1-dependent. The expression of both, N1 and N2, clearly occurs before PR1 gene expression. The analysis of GUS-reporter gene constructs confirms the early SA-dependent induction of the N1 and N2 promoters before activation of the NtPR1a promoter. Similar to the PR1a promoter, both NIMIN promoters can be induced by thiamine-HCl and show an inhibitory effect of the JA signaling network on the strength of reporter gene expression during simultaneous treatment with SA and JA. At the histological level, the N1 and N2 promoters display SA-dependent activation in leaf and root tissue of young tobacco seedlings. This activity clearly differs from the N3 promoter. The N2 promoter ? just like the AtPR1 and NtPR1a promoters ? contains an as-1-like element with a tandem repeat of TGACG, responsible for the SA sensitivity of the promoter. However the N2 as-1-like element structurally differs from the as-1-like elements in the PR1 promoters. In the N1 promoter a SA-responsive element was located in the region of -436 to -402 with respect to the translation starting point of the N1 gene. However, mutation of a TGATG repeat within this region did not result in a loss of promoter activity. Analysis of chimeric promoter constructs with foreign as-1-like elements showed that the different expression kinetics of PR1 and NIMIN genes are not encoded by the genetic information of the respective as-1-like elements. On the contrary, the as-1-like cis-acting elements affect the promoters? tissue specificity. Due to the integration of the N2-as-1-like element, the NtPR1a promoter, which is solely active in leaf tissue, adopts the typical NIMIN activity in root tissue. The presence of the 35S-as-1 element in the NtPR1a promoter leads to constitutional activation in root tissue. However, the activation in leaf tissue is still SA-dependent. In the yeast one-hybrid system, the interaction of TGA factors with as-1 and the as-1-like elements of the AtPR1, NtPR1a and N2 promoters shows only small differences in binding quality, whereas considerable differences can be detected in quantitative binding strength. Mutation of the as-1-like elements in the NtPR1a and N2 promoters results in the loss of TGA factor binding. The N1 promoter region from -436 to -399 contains a TGA binding site. Mutation of the contained TGATG repeat leads to a total loss of binding of TGA transcription factors. The neighbouring promoter context can exert both positive and negative influence on TGA factor binding. In case of the N1 promoter, the presence of adjacent promoter regions results in increased binding affinity of TGA factors. In contrast, additional NtPR1a promoter context shows a considerable reduction of TGA factor binding to as-1-like elements. Despite independent binding sites, NIMIN proteins and TGA transcription factors compete for binding of AtNPR1 in the yeast three-hybrid system. The presence of N1 and N3 thereby impedes interaction of TGA factors with NPR1, whereas simultaneous binding of N2 and TGA factor is possible without any restrictions. Binding of N1 or N2 simultaneously with N3 at the C- and N-termini of NPR1 also results in reciprocal interference suggesting a spatial folding of NPR1 where the N- and C-termini lie closely together.
Wirt-Virus Wechselwirkungen bei der Infektion durch Acanthocystis turfacea Chlorella Virus 1 : Regulation der Genexpression früher Gene und des Ubiquitin-Systems
(2021) Lindner, Kamila; Pfitzner, Artur J. P.
Acanthocystis turfacea Chlorella Virus 1 (ATCV-1) is a virus of the genus Chloroviruses that infects the unicellular green alga Chlorella heliozoae. The infection with ATCV-1 is lethal for the algae and requires the correct expression of the 860 hypothetical virus genes. These genes are divided into early, early/late and late genes which are expressed at different times, depending on their protein function. Gene expression is regulated by their corresponding viral promoters and can be controlled by viral or host-specific transcription factors. The stability of proteins is regulated by the host’s ubiquitin system. This study investigated the expression of early viral genes and the ubiquitin- mediated protein degradation regulated by ATCV-1. By investigating the promoters of the early genes of ATCV-1 Z174L, Z765R and Z798L respectively, consensus sequences were identified including a Hex motif and a TATA box that can be bound by viral, but most importanly, host transcription factors. By in vivo interactions with G-box binding factors, a direct regulation of the early promoters, including aforementioned Hex motif, could be demonstrated. Along with this new regulatory mechanism for the expression of early viral genes, evidence for additional mechanisms for the regulation of early genes with different consensus sequences, such as AATGACA, were found. In the second part of this study, three novel viral proteins were identified as proteins of the ubiquitin system: a viral ubiquitin (Z203L), a viral RING E3 ligase (Z292L) and a viral SKP1 protein (Z339L). Experiments have shown that ATCV-1 is able to interfere with the host’s ubiquitin system using these proteins. Although the E3 ligases are responsible for the specific ubiquitination of the target proteins (e.g. Z292L in the case of enolase), the additional expression of a viral ubiquitin ensures a sufficient amount of the signal protein is present. The results of this study demonstrate a wide range of host-virus interactions at the level of gene regulation and protein degradation. ATCV-1 can use host factors to initiate its own gene expression and, with the help of the components of the ubiquitin system encoded in the viral genome, reprogram the host’s protein degradation.