Browsing by Subject "Cell death"

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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.
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.