Browsing by Subject "SAR"

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    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.
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    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.
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    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.