TGA Transcription Factors Research Articles

Genetic Interactions of TGA Transcription Factors in the Regulation of Pathogenesis-Related Genes and Disease Resistance in Arabidopsis

TGA transcription factors are implicated as regulators of pathogenesis-related (PR) genes because of their physical interaction with the known positive regulator, nonexpresser of PR gene1 (NPR1). A triple-knockout mutant tga2-1 tga5-1 tga6-1 was shown previously to be defective in the induction of PR genes and systemic acquired resistance, confirming their role in disease resistance. However, the contributions of individual TGA factors have been difficult to discern because of functional redundancy among these factors, as well as possible dual functions for some single factors. In this study, we characterized six TGA factors by reverse genetics. We show that TGA3 is required for both basal and 2,6-dichloroisonicotinic acid-induced transcription of PR genes. The tga3-1 mutants were found to be defective in basal pathogen resistance, whereas induced resistance was unaffected. TGA1 and TGA4 play partially redundant roles in regulation of basal resistance, having only moderate effects on PR gene expression. Additionally, an activation-tagged mutant of TGA6 was able to increase basal as well as induced expression of PR1, demonstrating a positive role for TGA6 on PR gene expression. In contrast, TGA2 has repressor activity on PR gene expression even though it can act as a positive regulator in the tga5-1 tga6-1 null mutant background. Finally, we examined the genetic interaction between tga2-2 and suppressor of npr1 inducible1 (sni1-1). TGA2's repressor activity overlaps with SNI1 because the tga2-2 sni1-1 double mutant shows a synergistic effect on PR gene expression.

The Coactivator Function ofArabidopsisNPR1 Requires the Core of Its BTB/POZ Domain and the Oxidation of C-Terminal Cysteines

NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) regulates systemic acquired resistance (SAR) in Arabidopsis thaliana, and current models propose that after treatment with salicylic acid (SA), Cys-82 and Cys-216 of NPR1 are reduced, leading to nuclear import. The interaction of nucleus-localized NPR1 with TGA transcription factors results in the activation of defense genes, including the SAR marker PATHOGENESIS-RELATED-1 (PR-1), and the deployment of SAR. Little is known about how TGA factors or NPR1 regulate transcription or whether a TGA-NPR1 complex forms on DNA. We show that TGA2 and NPR1 are recruited to PR-1 independently of each other and of SA treatment. Consistent with the result that a triple knockout in TGA2/5/6 derepresses PR-1, in vivo plant transcription assays revealed that TGA2 is not an autonomous transcription activator but is a transcriptional repressor in both untreated and SA-treated cells. However, after stimulation with SA, TGA2 is incorporated into a transactivating complex with NPR1, forming an enhanceosome that requires the core of the NPR1 BTB/POZ domain (residues 80 to 91) and the oxidation of NPR1 Cys-521 and Cys-529. These Cys residues are found in a new type of transactivation domain that we term Cys-oxidized. These data further our understanding of the mechanism by which TGA2 and NPR1 activate Arabidopsis PR-1.

Fine-Tuning Plant Defence Signalling: Salicylate versus Jasmonate

Plant defences against pathogens and herbivorous insects form a comprehensive network of interacting signal transduction pathways. The signalling molecules salicylic acid (SA) and jasmonic acid (JA) play important roles in this network. SA is involved in signalling processes providing systemic acquired resistance (SAR), protecting the plant from further infection after an initial pathogen attack. SAR is long-lasting and provides broad spectrum resistance to biotrophic pathogens that feed on a living host cell. The regulatory protein NPR1 is a central positive regulator of SAR. SA-activated NPR1 localizes to the nucleus where it interacts with TGA transcription factors to induce the expression of a large set of pathogenesis-related proteins that contribute to the enhanced state of resistance. In a distinct signalling process, JA protects the plant from insect infestation and necrotrophic pathogens that kill the host cell before feeding. JA activates the regulatory protein COI1 that is part of the E3 ubiquitin ligase-containing complex SCFCOI1, which is thought to derepress JA-responsive genes involved in plant defence. Both synergistic and antagonistic interactions have been observed between SA- and JA-dependent defences. NPR1 has emerged as a critical modulator of cross-talk between the SA and JA signal and is thought to aid in fine tuning defence responses specific to the encountered attacker. Here we review SA- and JA-dependent signal transduction and summarize our current understanding of the molecular mechanisms of cross-talk between these defences.

BLADE-ON-PETIOLE–Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis

NONEXPRESSOR OF PR GENES1 (NPR1) is a key regulator of the plant defense response known as systemic acquired resistance. Accumulation of the signal molecule salicylic acid (SA) leads to a change in intracellular redox potential, enabling NPR1 to enter the nucleus and interact with TGACG sequence-specific binding protein (TGA) transcription factors, which in turn bind to SA-responsive elements in the promoters of defense genes. Here, we show that two NPR1-like genes, BLADE-ON-PETIOLE1 (BOP1) and BOP2, function redundantly to control growth asymmetry, an important aspect of patterning in leaves and flowers. Phenotypes in the double mutant include leafy petioles, loss of floral organ abscission, and asymmetric flowers subtended by a bract. We demonstrate that BOP2 is localized to both the nucleus and the cytoplasm, but unlike NPR1, it is highly expressed in young floral meristems and in yeast interacts preferentially with the TGA transcription factor encoded by PERIANTHIA (PAN). In support of a biological relevance for this interaction, we show that bop1 bop2 and pan mutants share a pentamerous arrangement of first whorl floral organs, a patterning defect that is retained in bop1 bop2 pan triple mutants. Our data provide evidence that BOP proteins control patterning via direct interactions with TGA transcription factors and demonstrate that a signaling mechanism similar to that formally associated with plant defense is likely used for the control of developmental patterning.

An Arabidopsis NPR1‐like gene, NPR4, is required for disease resistance

The Arabidopsis genome contains six NPR1-related genes. Given the pivotal role played by NPR1 in controlling salicylic acid (SA)-mediated gene expression and disease resistance, functional characterization of other family members appears to be justified. Reverse genetics was used to analyze the role of one NPR1-like gene, which we called NPR4. The NPR4 protein shares 36% identity with NPR1 and interacts with the same spectrum of TGA transcription factors in yeast two-hybrid assays. Plants with T-DNA insertions in NPR4 are more susceptible to the virulent bacterial pathogen Pseudomonas syringe pv. tomato DC3000. This phenotype is complemented by expression of the wild type NPR4 coding region. As determined by the parasite reproduction, the npr4-1 mutant is more susceptible to the fungal pathogen Erysiphe cichoracearum, but does not differ markedly from wild type in its interaction with virulent and avirulent strains of the oomycete Peronospora parasitica. In leaves of wild-type plants, NPR4 mRNA levels increase following pathogen challenge or SA treatment, and decrease rapidly following methyl jasmonic acid (MeJA) treatment. Transcripts of the pathogenesis-related (PR) genes PR-1, PR-2, and PR-5 are only marginally reduced in the npr4-1 mutant following pathogen challenge or SA treatment. This reduction of PR gene expression is more pronounced when leaves are challenged with the bacterial pathogen following SA treatment. Expression of the jasmonic acid-dependent pathway marker gene PDF1.2 is compromised in npr4-1 leaves following application of MeJA or a combination of SA and MeJA. These results indicate that NPR4 is required for basal defense against pathogens, and that it may be implicated in the cross-talk between the SA- and JA-dependent signaling pathways.

NPR1, all things considered.

Recent work has shown that the Arabidopsis NPR1 protein not only plays an essential role in salicylic acid (SA)-mediated systemic acquired resistance and rhizobacterium-triggered induced systemic resistance, but also is involved in crosstalk inhibition of jasmonic acid (JA)-mediated defense responses. Molecular characterization has revealed that activation of NPR1 and certain TGA transcription factors occurs under the reducing conditions that follow an initial oxidative burst after the induction of defense responses. In addition to NPR1 and TGA, the single-stranded DNA-binding transcription factor AtWhy1 and the WRKY70 transcription factor were recently found to be involved in SA-mediated defense and SA-JA crosstalk, respectively.

Two MAPK cascades, NPR1, and TGA transcription factors play a role in Pto-mediated disease resistance in tomato.

The tomato Pto kinase confers resistance to the causative agent of bacterial speck disease, Pseudomonas syringae pv. tomato, by recognizing the pathogen effector proteins AvrPto or AvrPtoB. Pto-mediated resistance requires multiple signal transduction pathways and has been shown to activate many defense responses including an oxidative burst, rapid changes in the expression of over 400 genes, and localized cell death. We have tested the role in Pto-mediated resistance in tomato of a set of 21 genes from other species known to be involved in defense-related signaling. Expression of each gene was suppressed by virus-induced gene silencing (VIGS) and the effect on disease symptoms and bacterial growth during the tomato-Pseudomonas incompatible interaction was determined. We found that Pto-mediated resistance was compromised by silencing of genes encoding two mitogen-activated protein (MAP) kinase kinases, MEK1 and MEK2, two MAP kinases, NTF6 and wound-induced protein kinase (WIPK), a key regulator of systemic acquired resistance (SAR), NPR1, and two transcription factors, TGA1a and TGA2.2. A lesser impact on Pto-mediated resistance was observed in plants silenced for RAR1 and COI1. The identification of nine genes that play a role in resistance to bacterial speck disease both advances our knowledge of Pto signal transduction and demonstrates the conservation of many defense signaling components among diverse plant species.

Knockout analysis of Arabidopsis transcription factors TGA2, TGA5, and TGA6 reveals their redundant and essential roles in systemic acquired resistance.

Arabidopsis nonexpresser of pathogenesis-related (PR) genes (NPR1) is the sole positive regulator that has been shown to be essential for the induction of systemic acquired resistance. In npr1 mutant plants, salicylic acid (SA)-mediated PR gene expression and pathogen resistance are abolished completely. NPR1 has been shown to interact with three closely related TGA transcription factors-TGA2, TGA5, and TGA6-in yeast two-hybrid assays. To elucidate the biological functions of these three TGA transcription factors, we analyzed single and combined deletion knockout mutants of TGA2, TGA5, and TGA6 for SA-induced PR gene expression and pathogen resistance. Induction of PR gene expression and pathogen resistance by the SA analog 2,6-dichloroisonicotinic acid (INA) was blocked in tga6-1 tga2-1 tga5-1 but not in tga6-1 or tga2-1 tga5-1 plants. Loss of INA-induced resistance to Peronospora parasitica Noco2 cosegregated with the tga6-1 mutation in progeny of multiple lines that were heterozygous for tga6-1 and homozygous for tga2-1 tga5-1 and could be complemented by genomic clones of wild-type TGA2 or TGA5, indicating that TGA2, TGA5, and TGA6 encode redundant and essential functions in the positive regulation of systemic acquired resistance. In addition, tga6-1 tga2-1 tga5-1 plants had reduced tolerance to high levels of SA and accumulated higher basal levels of PR-1 under noninducing conditions, suggesting that these TGA factors also are important for SA tolerance and the negative regulation of the basal expression of PR-1.

Systemic acquired resistance.

Systemic acquired resistance (SAR) is a mechanism of induced defense that confers long-lasting protection against a broad spectrum of microorganisms. SAR requires the signal molecule salicylic acid (SA) and is associated with accumulation of pathogenesis-related proteins, which are thought to contribute to resistance. Much progress has been made recently in elucidating the mechanism of SAR. Using the model plant Arabidopsis, it was discovered that the isochorismate pathway is the major source of SA during SAR. In response to SA, the positive regulator protein NPR1 moves to the nucleus where it interacts with TGA transcription factors to induce defense gene expression, thus activating SAR. Exciting new data suggest that the mobile signal for SAR might be a lipid molecule. We discuss the molecular and genetic data that have contributed to our understanding of SAR and present a model describing the sequence of events leading from initial infection to the induction of defense genes.

Differential regulation of TGA transcription factors by post-transcriptional control.

Transcription factors often belong to multigene families and their individual contribution in a particular regulatory network remains difficult to assess. We show here that specific members from a family of conserved Arabidopsis bZIP transcription factors, the TGA proteins, are regulated in their protein stability by developmental stage-specific proteolysis. Using GFP fusions of three different Arabidopsis TGA factors that represent members of distinct subclasses of the TGA factor family, we demonstrate that two of these TGA proteins are specifically targeted for proteolysis in mature leaf cells. Using a supershift gel mobility assay, we found evidence for similar regulation of the cognate proteins as compared to the GFP fusion proteins expressed under the cauliflower mosaic virus (CaMV) 35S promoter. Using various inhibitors, we showed that the expression of at least one of these three TGA factors could be stabilized by inhibition of proteasome-mediated proteolysis. This study indicates that TGA transcription factors may be regulated by distinct pathways of targeted proteolysis that can serve to modulate the contribution of specific members of a multigene family in complex regulatory pathways.

Abiotic and biotic stress differentially stimulate as-1 element activity in Arabidopsis

As-1-type cis-elements enhance the expression of putative plant protective genes in response to xenobiotic chemical stress. Here we investigate the effect of developmental changes on as-1-dependent transcription activities under basal and stimulus-induction conditions. We show that different xenobiotic stress cues give rise to distinct, differential patterns of as-1 activity in planta, which are further effected by developmental control. Moreover, transcription through as-1 is potentiated by infection with a virulent strain of Pseudomonas syringae, in line with recent genetic evidence indicating a role for this element and its cognate TGA transcription factors in host-defense gene responses. Our results here are consistent with the notion that inputs from both developmental and stimulus induction pathways define the complex spatial and temporal expression pattern of as-1 activity in plants.

Trans-dominant suppression of plant TGA factors reveals their negative and positive roles in plant defense responses.

Salicylic acid (SA) is a key regulator for the induction of systemic acquired resistance (SAR), and NPR1 is a critical mediator for the biological effects of SA. Physical interactions between NPR1 and TGA factors, a conserved family of basic-leucine-zipper (bZip) proteins in plants, have suggested a role for these transcription factors in mediating SAR induction via the regulation of defense genes. To elucidate this function, we constructed a trans-dominant mutant that specifically eliminates DNA-binding activities of this class of bZip proteins in transgenic tobacco plants. Our results demonstrate that the loss of TGA DNA-binding activities is correlated with suppression of two xenobiotic-responsive genes, GNT35 and STR246, and enhanced induction of pathogenesis-related (PR) genes by SA. In addition, these TGA-suppressed plants exhibited higher levels of PR gene induction by pathogen challenge and an enhanced SAR. These results suggest that TGA transcription factors serve both negative and positive regulatory roles in mediating plant defense responses.