Browsing by Subject "NIMIN"

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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.
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.
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.