Systemic acquired resistance (SAR) is a plant immune response established in uninfected leaves after colonization of local leaves with biotrophic or hemibiotrophic pathogens. The amino acid-derived ...metabolite N-hydroxypipecolic acid (NHP) travels from infected to systemic leaves, where it activates salicylic acid (SA) biosynthesis through the isochorismate pathway. The resulting increased SA levels are essential for induction of a large set of SAR marker genes and full SAR establishment. In this study, we show that pharmacological treatment of Arabidopsis thaliana with NHP induces a subset of SAR-related genes even in the SA induction-deficient2 (sid2/isochorismate synthase1) mutant, which is devoid of NHP-induced SA. NHP-mediated induction is abolished in sid2-1 NahG plants, in which basal SA levels are degraded. The SA receptor NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) and its interacting TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA) transcription factors are required for the NHP-mediated induction of SAR genes at resting SA levels. Isothermal titration analysis determined a KD of 7.9 ± 0.5 µM for the SA/NPR1 complex, suggesting that basal levels of SA would not bind to NPR1 unless yet unknown potentially NHP-induced processes increase the affinity. Moreover, the nucleocytoplasmic protein PHYTOALEXIN DEFICIENT4 is required for a slight NHP-mediated increase in NPR1 protein levels and NHP-induced expression of SAR-related genes. Our experiments have unraveled that NHP requires basal SA and components of the SA signaling pathway to induce SAR genes. Still, the mechanism of NHP perception remains enigmatic.
• TGACG-BINDING FACTORs (TGAs) control the developmental or defense-related processes. In Arabidopsis thaliana, the functions of at least TGA2 and PERIANTHIA (PAN) can be repressed by interacting ...with CC-type glutaredoxins, which have the potential to control the redox state of target proteins. As TGA1 can be redox modulated in planta, we analyzed whether some of the 21 CC-type glutaredoxins (ROXYs) encoded in the Arabidopsis genome can influence TGA1 activity in planta and whether the redox active cysteines of TGA1 are functionally important.
• We show that the tga1 tga4 mutant and plants ectopically expressing ROXY8 or ROXY9 are impaired in hyponastic growth. As expression of ROXY8 and ROXY9 is activated upon transfer of plants from hyponasty-inducing low light to normal light, they might interfere with the growth-promoting function of TGA1/TGA4 to facilitate reversal of hyponastic growth.
• The redox-sensitive cysteines of TGA1 are not required for induction or reversal of hyponastic growth.
• TGA1 and TGA4 interact with ROXYs 8, 9, 18, and 19/GRX480, but ectopically expressed ROXY18 and ROXY19/GRX480 do not interfere with hyponastic growth. Our results therefore demonstrate functional specificities of individual ROXYs for distinct TGAs despite promiscuous protein–protein interactions and point to different repression mechanisms, depending on the TGA/ROXY combination.
Summary
A cauliflower mosaic virus (CaMV) 35S promoter derivative, which is tightly repressed by the Tn 10 encoded Tet repressor in a transient expression system as well as in transgenic plants has ...been constructed. After treatment of transgenic plants with tetracycline (Tc) the activity of the reporter enzyme β‐glucuronidase (GUS) increased up to 500‐fold in tissue culture as well as under greenhouse conditions. Efficient derepression was achieved by Tc uptake through the roots as well as by Tc treatment of leaves of intact plants. As Tc is not very stable in the plants, this system can also be used for a transient expression of a transgene. This system provides a unique tool for regenerating transgenic plants carrying a repressed transgene and for efficiently de‐repressing its activity by a specific inducer at any time point of further development.
Summary
Salicylic acid (SA) is a plant signaling molecule that mediates the induction of defense responses upon attack by a variety of pathogens. Moreover, it antagonizes gene induction by the stress ...signaling molecule jasmonic acid (JA). Several SA‐responsive genes are regulated by basic/leucine zipper‐type transcription factors of the TGA family. TGA factors interact with NPR1, a central regulator of many SA‐induced defense responses including SA/JA antagonism. In order to identify further regulatory proteins of SA‐dependent signaling pathways, a yeast protein interaction screen with tobacco TGA2.2 as bait and an Arabidopsis thaliana cDNA prey library was performed and led to the identification of a member of the glutaredoxin family (GRX480, encoded by At1g28480). Glutaredoxins are candidates for mediating redox regulation of proteins because of their capacity to catalyze disulfide transitions. This agrees with previous findings that the redox state of both TGA1 and NPR1 changes under inducing conditions. Transgenic Arabidopsis plants ectopically expressing GRX480 show near wild‐type expression of standard marker genes for SA‐ and xenobiotic‐inducible responses. In contrast, transcription of the JA‐dependent defensin gene PDF1.2 was antagonized by transgenic GRX480. This, together with the observation that GRX480 transcription is SA‐inducible and requires NPR1, suggests a role of GRX480 in SA/JA cross‐talk. Suppression of PDF1.2 by GRX480 depends on the presence of TGA factors, indicating that the GRX480/TGA interaction is effective in planta.
Summary
A chemically regulated gene expression system that can be switched on with dexamethasone and switched off with tetracycline was constructed. It is based on a transcriptional activator (TGV) ...that consists of the Tn10 encoded Tet repressor, the rat glucocorticoid receptor hormone binding domain and the transcriptional activation domain of Herpes simplex virion protein VP16. When stably expressed in transgenic tobacco plants, it mediates dexamethasone‐inducible transcription from a synthetic promoter (PTop10) consisting of seven tet operators upstream of a TATA‐box. Tetracycline interferes with induction by negatively regulating the DNA‐binding activity of the TetR moiety of TGV. The boundaries of the expression window of the TGV‐driven PTop10 reach from undetectable levels of the reporter enzyme β‐glucuronidase in the absence of dexa‐ methasone to induced levels reaching 15–20% of the Cauliflower Mosaic Virus 35S promoter (PCaMV35S). By modifying the sequence of PTop10, we generated a new target promoter (PTax) that is stably expressed over several generations and that can be activated to levels comparable to PCaMV35S, while yielding only slightly elevated background activities.
Glutaredoxins (GRXs) are small proteins which bind glutathione to either reduce disulfide bonds or to coordinate iron sulfur clusters. Whereas these well-established functions are associated with ...ubiquitously occurring GRXs that encode variants of a CPYC or a CGFS motif in the active center, land plants also possess CCxC/S-type GRXs (named ROXYs in Arabidopsis thaliana) for which the biochemical functions are yet unknown. ROXYs and CC-type GRXs from rice and maize physically and genetically interact with bZIP transcription factors of the TGA family to control developmental and stress-associated processes. Here we demonstrate that ROXYs interact with transcriptional co-repressors of the TOPLESS (TPL) family which are related to Tup1 in fungi and Groucho/TLE in animals. In ROXYs, the functionally important conserved A(L/I)W(L/V) motif at the very C terminus mediates the interaction with TPL. A ternary TGA2/ROXY19/TPL complex is formed when all three proteins are co-expressed in yeast. Loss-of-function evidence for the role of TPL in ROXY19-mediated repression was hampered by the redundancy of the five members of the TPL gene family and developmental defects of higher order tpl mutants. As an alternative strategy, we ectopically expressed known TPL-interacting proteins in order to out-compete the amount of available TPL in transiently transformed protoplasts. Indeed, ROXY19-mediated transcriptional repression was strongly alleviated by this approach. Our data suggest a yet unrecognized function of GRXs acting as adapter proteins for the assembly of transcriptional repressor complexes on TGA-regulated target promoters.
•Glutaredoxin ROXY19 interacts simultaneously with TGA transcription factors and with the co-repressor TOPLESS.•Mutating the conserved ALWL motif into an ALWA motif renders ROXY1 and ROXY19 unable to interact with TOPLESS.•Mutating the ALWL motif into an ALWA motif renders ROXY19 unable to repress transcription.•Proteins that compete with ROXY19 for binding to TOPLESS interfere with the repressive function of ROXY19.•Our conclusion: ROXYs repress transcription by bridging TGA factors with TOPLESS at specific target promoters.
Two antagonistic hormonal pathways merge at the promoter of a crucial activator of one of the pathways.
Salicylic acid (
SA
), a hormone essential for defense against biotrophic pathogens, triggers ...increased susceptibility of plants against necrotrophic attackers by suppressing the jasmonic acid-ethylene (
ET
) defense response. Here, we show that this disease-promoting
SA
effect is abolished in plants lacking the three related TGACG sequence-specific binding proteins TGA2, TGA5, and TGA6 (class II TGAs). After treatment of plants with the
ET
precursor 1-aminocyclopropane-1-carboxylic acid (
ACC
), activation of all those genes that are suppressed by
SA
depended on class II TGAs. Rather than TGA binding sites, GCC-box motifs were significantly enriched in the corresponding promoters. GCC-box motifs are recognized by members of the superfamily of APETALA2/ETHYLENE RESPONSE FACTORs (ERFs). Of 11 activating
ACC
-induced
APETALA2/ERFs
, only
ORA59
(for
OCTADECANOID-RESPONSIVE ARABIDOPSIS APETALA2/ETHYLENE RESPONSE FACTOR domain protein59
) and
ERF96
were strongly suppressed by
SA
. ORA59 is the master regulator of the jasmonic acid-
ET
-induced defense program.
ORA59
transcript levels do not reach maximal levels in the
tga2 tga5 tga6
triple mutant, and this residual activity cannot be suppressed by
SA
. The
ORA59
promoter contains an essential TGA binding site and is a direct target of class II TGAs as revealed by chromatin immunoprecipitation experiments. We suggest that class II TGAs at the
ORA59
promoter constitute an important regulatory hub for the activation and
SA
suppression of
ACC
-induced genes.
Verticillium longisporum is a soil-borne vascular pathogen that causes reduced shoot growth and early senescence in Arabidopsis (Arabidopsis thaliana). Here, we report that these disease symptoms are ...less pronounced in plants that lack the receptor of the plant defense hormone jasmonic acid (JA), CORONATINE INSENSITIVE1 (COI1). Initial colonization of the roots was comparable in wild-type and coil plants, and fungal DNA accumulated to almost similar levels in petioles of wild-type and coil plants at 10 d post infection. Completion of the fungal life cycle was impaired in coil, as indicated by the reduced number of plants with microsclerotia, which are detected on dead plant material at late stages of the disease. Contrary to the expectation that the hormone receptor mutant coi1 should display the same phenotype as the corresponding hormone biosynthesis mutant delayed dehiscence2 (dde2), dde2 plants developed wild-type-like disease symptoms. Marker genes of the JA and the JA/ethylene defense pathway were induced in petioles of wild-type plants but not in petioles of dde2 plants, indicating that fungal compounds that would activate the known COI1-dependent signal transduction chain were absent. Grafting experiments revealed that the susceptibility-enhancing COI1 function acts in the roots. Moreover, we show that the coi1 -mediated tolerance is not due to the hyperactivation of the salicylic acid pathway. Together, our results have unraveled a novel COI1 function in the roots that acts independently from JA-isoleucine or any JA-isoleucine mimic. This COI1 activity is required for a yet unknown root-to-shoot signaling process that enables V. longisporum to elicit disease symptoms in Arabidopsis.
The chimeric transcriptional activator tTA, a fusion between the Tn10 encoded Tet repressor and the activation domain of the Herpes simplex virion protein VP16, was stably expressed in transgenic ...tobacco plants. It stimulates transcription of the beta-glucuronidase (gus) gene from an artificial promoter consisting of 7 tet operators and a TATA-box. Tetracycline, which interferes with binding of tTA to operator DNA, reduces gus expression over several orders of magnitude. This stringency of regulation suggests that the system can be used to construct transgenic plants encoding a potentially lethal gene product. Furthermore, the specific and fast inactivation of tTA allows study of the stability of RNAs and proteins.
For understanding the mechanism of transcriptional regulation, it is essential to know which transcription factor is bound in vivo to the promoter to be analysed. If transcription from a given ...promoter is regulated by developmental or environmental stimuli, the question of inducible versus constitutive binding has to be answered, particularly if the transcriptional regulator is expressed both under uninduced and induced conditions. Chromatin immunoprecipitation (ChIP) assays constitute the most adequate approach to address these issues as proteins are cross-linked to the DNA before disruption of the tissue. Thus, the DNA-protein interaction is stabilized during purification of the chromatin. The specific DNA-protein complex is immuno-enriched employing specific antibodies against the transcription factor to be analysed. After reversal of cross-links, the recovered DNA is amplified by PCR using specific primers that match sequences flanking the suspected binding site. The amount of PCR product is indicative of the relative abundance of the DNA-protein complex in vivo. A protocol for ChIP assays for Arabidopsis thaliana leaves is described.
PDF
