Characterization of an Arabidopsis glutaredoxin that interacts with core components of the salicylic acid signal transduction pathway

Abstract

Salicylic acid (SA) is a plant signalling molecule that mediates the induction of defense responses upon attack by a variety of pathogens. Activation sequence-1 (as-1) type cis elements and their cognate basic/leucine zipper (bZIP)-type transcription factors of the TGA family regulate transcription in response to SA and in response to xenobiotic chemicals. TGA factors interact with NPR1 (NON EXPRESSOR OF PR GENES 1), a central regulator of many SA-induced defense responses. Changes in the redox state of both TGA1 and NPR1 have been observed under inducing conditions. In addition to induction of SA-inducible genes, NPR1 is also involved in suppression of jasmonic acid (JA)-inducible genes under conditions of the simultaneous presence of increased level of SA and JA in the cell.A yeast protein interaction screen with tobacco NtTGA2.2 as a bait and an Arabidopsis thaliana cDNA prey library had previously identified a member of the glutaredoxin family (GRX480, encoded by At1g28480) as a TGA interacting protein. Glutaredoxins are candidates for mediating redox regulation of proteins because of their capacity to catalyze disulfide transitions. GRX480 is localized in the cytosol and the nucleus of plant protoplasts. A ternary GRX480/NtTGA2.2/NPR1 complex could be detected in the yeast three hybrid assay.In this study, we used the yeast and plant protoplast two-hybrid assays to assess the influence of the two catalytic cysteines of GRX480 on the interaction with AtTGA2. Individual yeast clones showed a considerable variability with respect to the interaction that did not correlate with the expression of the interacting proteins. Nevertheless it can be concluded that the redox-deficient GRX480 still interacts with AtTGA2 in yeast. This interaction seems to be compromised in plant protoplasts. Furthermore, the interaction does not depend on the GRX480-specific N terminus. AtTGA2 does not interact with the related glutaredoxin GRX370 (At5g40370).GRX480 is expressed in response to SA and pathogen challenge. SA-induced expression depends on NPR1 and TGA factors. Though the gene is not inducible by JA, JA can enhance SA-induced expression by a factor of 2.Arabidopsis lines ectopically expressing GRX480 show reduced transcriptional activation from the truncated CaMV 35S promoter that contains the as-1 element as the only cis regulatory element. This was not observed upon ectopic expression of GRX370 indicating that the interaction with TGA factors might be important for this effect.The JA-responsive defensin gene PDF1.2, that is subject to the SA/JA antagonism, is also negatively regulated by GRX480, suggesting that GRX480 is a regulatory component of this cross-talk. GRX480 with cysteine to serine exchanges in the active centre did not mediate this effect. Epistasis analysis showed that GRX480 functions independently or downstream of NPR1. The functionality of the SA/JA antagonism in grx480 knock out plants suggests that redundant mechanisms exist in planta that lead to the strong suppression of PDF1.2 expression in Arabidopsis.