NPR1-DEPENDENT SA SIGNALING
To identify components involved in SA signal transduction, a number of mutant screens were performed that identified multiple alleles of a single gene, NPR1/NIM1. Further characterization showed that the role of NPR1 is not limited to SAR. The npr1 mutant also displays enhanced disease symptoms when infected with virulent pathogens and is impaired in some R gene-mediated resistance, suggesting that NPR1 is important for restricting the growth of pathogens at the site of infection. NPR1 is required for another induced resistance response, known as induced systemic resistance (ISR), which is triggered by nonpathogenic root-colonizing bacteria and confers resistance to bacteria and fungi in aerial parts of the plant. NPR1 also mediates cross-talk between the SA signaling pathway and the jasmonic acid (JA) and ethylene (C2H4) signaling pathways that confer resistance to insects and some necrotrophic pathogens. As discussed earlier, npr1 has reduced tolerance to SA toxicity and accumulates high endogenous levels of SA, suggesting a role for NPR1 in both detoxification of SA and feedback regulation of SA biosynthesis. In addition, NPR1 has functions that are not directly related to resistance, including the regulation of cell division and/or endoreduplication.
NPR1 is expressed throughout the plant at low levels and its mRNA levels rise two- to threefold after pathogen infection or treatment with SA. NPR1 expression is likely mediated by WRKY transcription factors as mutation of the WRKY binding sites (W-boxes) in the NPR1 promoter abolished its expression. Overexpression of NPR1 in Arabidopsis enhances resistance to P. parasitica, P. syringae, and Erysiphe cichoracearumwith no apparent detrimental effects on the plant. Unlike many mutants with constitutive resistance, the NPR1 overexpressing lines do not constitutively express PRgenes. The enhancement of resistance is probably caused by the more rapid or higher induction of PR genes observed in these overexpressing lines. This indicates that, even when expressed at higher levels, the NPR1 protein must be activated to induce SAR.
The NPR1 protein has two protein-protein interaction domains, an ankyrin-repeat and a BTB/POZ (Broad-Complex, Tramtrack, Bric-a-brac/Poxvirus, Zinc finger) domain, as well as a putative nuclear localization signal and phosphorylation sites. Functional studies have shown that accumulation of NPR1 in the nucleus after treatment with SAR inducers is essential for PR gene induction.
TGA Transcription Factors
The absence of any obvious DNA-binding domain and the presence of protein-protein interaction domains in NPR1 prompted several laboratories to carry out yeast two-hybrid screens for NPR1-interacting proteins. In one of these screens, three small structurally similar proteins named NIMIN1, NIMIN2, and NIMIN3 (NIM interactor) were identified. NIMIN1 and NIMIN2 interact with the C terminus of NPR1, while NIMIN3 interacts with the N terminus. NIMINs contain stretches of acidic amino acids and are hypothesized to be transcription factors; however, more experiments are required to demonstrate their biological activity.
The predominant NPR1 interactors found in the yeast two-hybrid screens were members of the TGA family of basic leucine zipper transcription factors. NPR1 interacts with the Arabidopsis TGA factors, TGA2, TGA3, TGA5, TGA6, and TGA7 but only weakly or not at all with TGA1 and TGA4. NPR1 also interacts with TGA factors from tobacco and rice. Using truncated or mutant forms of NPR1, the ankyrin-repeat domain in the middle of the protein was shown to be essential for binding TGA factors, while the N-terminal region appears to enhance binding.
TGA factors bind to activator sequence-1 (as-1) or as-1-like promoter elements, which have been found in several plant promoters activated during defense, including Arabidopsis PR-1. Linker scanning mutagenesis of the PR-1 promoter identified two as-1-like elements, LS7 and LS5. LS7 is a positive regulatory element required for induction by INA, whereas LS5 is a weak negative regulatory element. Després et al. used these cis-elements as probes for electrophoretic mobility shift assays (EMSA) and showed that both TGA2 and TGA4 could bind to LS7, whereas only TGA2 could bind to LS5. Furthermore, binding of TGA2 but not TGA4 was enhanced by the addition of NPR1, consistent with the yeast two-hybrid interaction data.
Although NPR1 is clearly a positive regulator of PR genes, it may exert its function by either enhancing a transcriptional activator or inhibiting a transcriptional repressor. The presence of multiple as-1-like elements in the PR-1 promoter and the differential binding affinities of each TGA factor to these elements as well as to NPR1 highlight the complexity of the regulatory mechanism. Indeed, in an EMSA performed by Després et al., binding to the as-1 element from the 35S promoter was significantly enhanced in protein extracts from SA-treated plants. However, extracts from untreated npr1plants also contained strong as-1-binding activity and this was not changed by SA treatment. This result can be reconciled if different TGA factors are responsible for the observed as-1-binding in wild type and npr1. Consistent with this hypothesis, when EMSA was performed using the LS7 element from the PR-1 promoter, Johnson et al. found that SA enhanced the binding activity in wild-type plants and this binding was abolished in npr1 plants. This result was confirmed by chromatin immunoprecipitation (ChIP) experiments. Recruitment of TGA2 or TGA3 to a 1kb fragment of the PR-1 promoter was observed only after treatment with SA and not in untreated wild-type plants or SA-treated npr1 plants. Unfortunately, ChIP is unable to resolve the adjacent binding sites LS7 and LS5, which are within 30 bp of each other.
Redox Signaling
The in vivo interaction of NPR1 with TGA factors requires induction with SA, even though both proteins are constitutively expressed. Until recently, the controlling mechanisms for NPR1 nuclear localization and activation of TGA factors were unclear. Two exciting new papers have revealed that changes in the redox status of the cell after SA treatment play an important role in this regulation.
The observation that NPR1-like proteins from different species contain ten conserved cysteines suggested that NPR1 might be under redox-regulation. Various researchers tested this hypothesis by examining NPR1 under different redox conditions. When proteins were extracted in the absence of the reducing agent DTT, monomeric NPR1 could only be detected in INA-treated samples, whereas in the presence of DTT equal amounts of monomeric NPR1 were detected with or without INA treatment. This suggests that before and after SAR induction NPR1 is present in two different conformations. As antibodies developed against the endogenous NPR1 only recognize the reduced form, further analyses used GFP-tagged NPR1. In extracts from untreated plants, NPR1-GFP was found in a high MW (>250,000) protein complex. Addition of DTT to the sample reduced all the NPR1-GFP to its monomeric form, suggesting that the high MW complex is formed through intermolecular disulfide bridges. Interestingly, this complex is partially reduced as a result of INA treatment. This pattern of conformational changes was also observed after pathogen infection in both inoculated and distal tissues and precedes PR gene induction. Measurements of cellular glutathione pools showed that a biphasic change in redox occurs after SAR induction, first oxidizing and then reducing. Lowering NADPH levels diminished both NPR1-GFP reduction and PR-1 expression after INA treatment. Mutation of cysteine residues C82 or C216 in NPR1 resulted in constitutive monomerization, nuclear accumulation of mutant proteins and expression of PR-1. These data demonstrate that the monomer is the active form of NPR1 for PR-1 induction and provide a model for the activation of NPR1 by SA: SA accumulation triggers conversion of NPR1 from an oligomer to a monomer through changes in cellular redox status favoring reduction. This monomeric form of NPR1 is then able to move to the nucleus where it interacts with TGA factors to induce PR gene expression.
As discussed earlier, in yeast two-hybrid studies NPR1 interacts strongly with TGA2 and TGA3 but very weakly or not at all with TGA1 and TGA. Using a plant two-hybrid assay in Arabidopsis, Després et al. demonstrated a physical interaction between NPR1 and TGA1. Using domain swapping between TGA1 and TGA2, the plant-specific regulatory region was defined to a 30 aa region containing two cysteine residues in TGA1 (and TGA4) that are not found in TGA2 or other TGA factors. Mutation of these residues in TGA1 allowed interaction with NPR1 in yeast and in untreated leaves. A clever labeling experiment designed to distinguish between reduced and oxidized cysteine residues showed that TGA1 (and/or TGA4) exists in both oxidized and reduced forms in untreated leaves. After SA treatment, only the reduced form was detected. This indicates that SA controls the redox status of TGA1 (and/or TGA4) and only the reduced form can bind NPR1. Evidently, redox changes do not increase TGA1 DNA-binding activity directly; instead, this is achieved through enhancement of its interaction with NPR1. These experiments add TGA1 and TGA4 to the spectrum of TGA factors that interact with NPR1, although their role in SAR has yet to be established. These results are consistent with the finding by Mou et al. that the SA signal is transduced through changes in cellular reduction potential that lead to monomerization of NPR. We are just beginning to realize the significance of such redox changes during SAR. In the past, research has mainly focused on the initial oxidative burst during R gene-mediated defense. These two studies highlight the importance of the reducing state that usually appears following the oxidative stress.