Browsing by Subject "NPR-Proteine"

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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.