Arabidopsis thaliana class II TGA transcription factors provide a molecular link between salicylic acid and ethylene defense signalling

Abstract

Since plants are exposed to a multitude of different attackers, a complex innate immune system has evolved to protect them from devastating diseases. Depending on the type of pathogen or pest, at least one the three major stress signalling hormones which coordinate further defense responses is synthesized (Glazebrook, 2005): Salicylic acid (SA) requires the redox-regulated co-activator NPR1 and TGA bZIP transcription factors to induce defense genes, is required to combat biotrophic pathogens; jasmonic acid (JA) leads to the degradation of JAZ repressor proteins to release MYC transcription factors; ethylene (ET) leads to the stabilization of transcription factor EIN3. Both pathways merge at the promoter of transcription factor ORA59 which triggers defense responses against necrotrophic pathogens (Pre et al., 2008). In the absence of ET, JA-activated MYC2 coordinates the response against insects. The three defense pathways act mutually antagonistic indicating that their simultaneous activation is evolutionary disadvantageous. This thesis has identified subclass II TGA transcription factors and their interacting CC-type glutaredoxins as the key regulatory module that mediates the antagonistic action of SA and JA on the JA/ET-pathway. In Chapter II we demonstrate that the Arabidopsis thaliana subclass II TGA transcription factors, which had been previously described as essential activators of the SA pathway, are positive regulators the JA/ET pathway. Evidence for this was provided by the increased susceptibility of tga256 triple mutant plants against the necrotrophic fungus Botrytis cinerea and decreased expression of the marker gene of the JA/ET response PDF1.2. In contrast, mutations in AtMYC2, the key positive regulator of the JA pathway, led to hyper-induction of the pathway. JA/ET-induced expression of PDF1.2 was restored in the tga256 myc2 quadruple mutant, indicating that TGA factors and MYC2 act as mutual suppressors on the JA/ET pathway. Interestingly, this tga256 myc2 mutant is insensitive to the antagonistic effect of SA establishing the concept that the positive function of TGA factors in the JA/ET-pathway serves to install the SA sensitivity. In Chapter III we show that the compromised defense gene expression in tga256 mutant plants after ET treatment is a direct consequence of the reduced expression of ORA59, the master integrator of the JA/ET pathway (Pre et al., 2008). Chromatin immunoprecipitation analyses demonstrated ET-induced direct binding of TGA factors to the TGACG motif at the ORA59 promoter. The functional importance of the TGA binding was further supported by analyses of transgenic ORA59Pro:GUS plants which indicated a crucial function of the TGACG motif for promoter activity. Moreover, SA-induced susceptibility of Arabidopsis plants towards infection with Botrytis cinerea was abolished in the tga256 mutant. Microarray analyses demonstrated that one third of all ET-induced genes is under positive transcriptional control of TGA factors. Interestingly, nearly all genes which are negatively affected by SA require TGA factors for being induced by ET. Collectively, these data support the idea that the positive function of TGA factors within the JA/ET pathway is targeted by SA to down-regulate the JA/ET-pathway. Chapter IV takes up the question how the antagonistic affect of SA on the TGA function is executed. A likely candidate is the previously described glutaredoxin GRX480 which combines multiple criteria of a cross-talk mediator: It interacts with TGA factors, its expression is SA-inducible and its over-expression suppresses JA/ET-induced defense gene expression (Ndamukong et al., 2007). This list was extended by our findings that the GRX480-mediated suppression is integrated at the ORA59 promoter. Loss-of-function evidence could not be provided, probably due to a functional redundancy within the glutaredoxin family. Seventeen plant-specific CC-type glutaredoxins were screened for their potential to suppress the ORA59 promoter using a transient expressions system that allowed monitoring the negative effect of glutaredoxins on the ORA59 promoter. Ten out of the 17 tested glutaredoxins revealed suppression capacity. Only these glutaredoxins contained the C-terminal ALWL motif which was previously described as crucial for glutaredoxins to mediate developmental processes in flowers (Li et al., 2009). In Chapter V, the interplay between MYC2 and GRX480 was explored since both factors negatively regulate the JA/ET-pathway in a TGA-dependent manner. JA-induced GRX480 expression was shown to be MYC2-dependent, giving rise to the hypothesis that MYC2 exerts its negative effect through GRX480. The down-regulation of the JA/ET-pathway as a result of the MYC2/GRX480 action removes the suppressive effect of the JA/ET-pathway on the JA-pathway. The hyper-stimulation of MYC2 expression which is observed in plants ectopically expressing GRX480, can therefore be regarded as a result of a MYC2-driven feed-forward loop. Collectively, the thesis has established a working model that envisions the ORA59 promoter as a target site for the antagonism of SA and JA on the JA/ET pathway. Mechanistically, this antagonism is established through TGA factors that enhance ORA59 promoter activity by synergistically interacting with EIN3 and a yet unknown JA-responsive transcription factor. TGA factors recruit JA- and SA-induced glutaredoxins that down-regulate ORA59 promoter activity by redox-modification of a yet unknown target protein.