Las Relaciones con el Entorno Social y Cultural

PR-1 gene activation is a molecular marker for the induction of SAR. The expression of this locus is controlled through the concerted efforts of the coactivator NPR1 and the functionally redundant TGA2-containing clade (TGA2, TGA5 and TGA6) of transcription factors. In the present paradigm for PR-gene expression, NPR1 is recognized as the key positive regulator of PR-1 induction. However, the TGA2 transcription factor plays an essential role in both the activation of this locus following stimulation with SA and its basal repression in resting cells. The duality demonstrated by TGA2 is critical to the regulation of PR-1 under both resting and inducing conditions.

In resting cells, the PR-1 gene is maintained in a repressed state by the TGA2-clade of transcription factors (Zhang et al., 2003; Rochon et al., 2006).

A role in PR-1 gene repression for the TGA2 transcription factor was first indicated when elevated levels of PR-1 expression were observed in the tga2/5/6 triple-knockout Arabidopsis mutant under non-inducing conditions (Zhang et al., 2003). The repression function of TGA2 was definitively demonstrated through the use of an in planta transcription assay (Rochon et al., 2006). This system showed that TGA2 could repress an activated reporter gene through the heterologous GAL4 DNA-binding domain. This system was further used to demonstrate that the native TGA2 factor could also repress reporter gene expression in the context of the PR-1 promoter (Rochon et al., 2006). These data suggest that the conformation adopted by the factor upon binding its cognate cis-element through its endogenous DNA-binding (DB) domain or that produced upon recruitment to the GAL4 upstream activating sequence (UAS) through the heterologous GAL4 DB domain are both sufficient to mediate the active repression of the reporter gene. However, it is not known if this repression is conducted by way of a conserved mechanism.

Linker scanning (LS) mutagenesis of the PR-1 promoter identified the presence of both positive and negative cis-regulatory elements (Lebel et al., 1998). TGA2 has been shown to bind the LS5 and LS7 promoter elements in vitro (Després et al., 2000). The LS5 appears to contribute to the negative regulation of PR-1 expression both in the absence and in the presence of SA, whereas LS7 is required for SA-mediated induction of PR-1 (Lebel et al., 1998). Chromatin immunoprecipitation (ChIP) studies have demonstrated that TGA2 is recruited to the PR-1 promoter in resting cells and notably this recruitment does not require NPR1 (Rochon et al., 2006). Due to limitations in resolution in the ChIP technique, these studies provided no indication as to which of the PR-1 promoter cis-regulatory elements is occupied by the transcription factor.

88 P. Boyle et al.

In resting cells, the NPR1 protein is localized to both the nucleus and the cytosol (Després et al., 2000). Through the use of the ChIP technique, it was revealed that the NPR1 coactivator is specifically present in the regulatory region of the PR-1 gene under non-inducing conditions, and it further demonstrated that the recruitment of the coactivator to this locus is independent of the TGA2 clade of transcription factors (Rochon et al., 2006). NPR1, like most coactivators, lacks a known DNA-binding domain and is therefore likely to be maintained at the repressed PR-1 promoter by way of another protein.

However, there is currently no information as to what the NPR1-anchoring entity might be, nor is there any indication of the function of NPR1 in this situation. The presence of other coactivators at unactivated or repressed promoters, although uncommon, has also been reported in Drosophila using the ChIP technique (Martinez and Arnosti, 2008). The presence of coactivators such as NPR1 at repressed promoters does not conform to the existing paradigm for gene regulation. However, it could be reasoned that the proximity of these latent coactivators to cis-regulatory elements renders them perfectly poised to activate gene expression in response to the appropriate cue. Despite the presence of NPR1 at the PR-1 promoter in resting cells, npr1 mutations do not affect the basal repression of the locus. Only the tga2/5/6 triple-knockout mutant demonstrates derepression of the PR-1 gene under non-inducing conditions (Zhang et al., 2003).

The requirement of NPR1 for the induction of SAR and the activation of PR-1 in response to SA is well documented by numerous different genetic screens (Cao et al., 1994; Delaney et al., 1995; Zhang et al., 2003). However, both the deployment of SAR and the expression of PR-1 also require the TGA2 clade of transcription factors. The necessity of these transcription factors in the activation of PR-1 and the deployment of SAR was not identified in the genetic screens due to the functional redundancy between TGA2, TGA5 and TGA6. The critical role of these factors in PR-1 regulation is in many ways undersold by their redundancy. The ability of the TGA transcription factors to interact with NPR1, both in the nucleus and in vitro, suggested a role in PR-1 activation (Després et al., 2000; Fan and Dong, 2002). However, the specific requirement of the TGA2 clade of factors in the activation of PR-1 was not appreciated until the development of the tga2/5/6 triple-knockout mutant (Zhang et al., 2003).

In SA-stimulated cells, like in resting cells, TGA2 is recruited to the PR-1 promoter in an NPR1-independent manner. The condition-invariant binding of the PR-1 regulatory region demonstrated by TGA2 is reminiscent of a behaviour that is also exhibited by another bZIP transcription factor, HY5 (Lee et al., 2007). ChIP studies have shown that HY5, the key positive regulator of photomorphogenesis, is constitutively bound to a multitude of light-induced loci and its recruitment is not affected by light conditions or light-to-dark transitions (Lee et al., 2007).

Although TGA2 and NPR1 are both present in the nucleus at the PR-1 promoter before SA treatment, the plant two-hybrid assay demonstrated that these factors do not interact until after stimulation with SA (Rochon et al., 2006). In the current model for PR-1 activation following stimulation with SA,

Disease Resistance in Arabidopsis: TGA2 and NPR1 89 NPR1 is incorporated into a transactivating complex with the TGA2 transcription factor, which nucleates the formation of an enhanceosome at the PR-1 promoter. It is important to note that the capacity for TGA2 to mediate repression is not directly affected by treatment with SA. This property of TGA2 was demonstrated through the use of an in planta transcription assay. In the absence of a functional NPR1, TGA2 continued to repress in the context of both the heterologous GAL4 UAS and PR-1 promoters in SA-stimulated cells (Rochon et al., 2006). Such an observation casts some doubts on the possibility that the ability of TGA2 to activate or repress transcription is modulated by a simple switch-type mechanism mediated by a post-translational modification stimulated by SA treatment. Further supporting this viewpoint is a previous study that demonstrated that the TGA2 transcription factor is phosphorylated by a casein kinase (CK)2-type kinase activity, which emerges following SA treatment. However, mutation of the phosphorylated residues in TGA2 did not affect the ability of the factor to activate PR-1 expression in response to SA stimulation (Kang and Klessig, 2005). The activator function of TGA2 is only realized when complexed with NPR1. However, the TGA2 activator function is not confined solely to the PR-1 promoter, since TGA2 can also activate gene expression in a heterologous context through the GAL4 DB in an SA- and NPR1-dependent manner (Rochon et al., 2006). The ability of TGA2 to manifest repressor/activator duality in these two unrelated contexts might suggest that the function of TGA2 is modulated minimally through DNA-binding allosteric effects. It is quite possible that the DNA DNA-binding mediated through its endogenous DNA-binding domain or by way of the GAL4 DB domain produces equivalent changes in the NPR1–TGA2 complex conforma-tion, resulting in a common means of activation. However, it is not possible to rule out the possibility that the complex adopts different conformations in these two contexts and that activation proceeds through different mechanisms. In this case, despite the dramatic difference in conformation changes imposed by the binding of different cis-elements, TGA2 would still be able to maintain the surfaces required to mediate repression and those necessary to recruit NPR1, ultimately effecting activation (Latchman, 2001; Natoli, 2004; Ma, 2005).

The ability of TGA2 to maintain these interfaces in different contexts would enable the factor to retain its transcriptional duality.

The NPR1 coactivator is constitutively present at the PR-1 promoter.

However, it appears to only activate PR-1 gene expression in SA-stimulated cells and requires the TGA2 clade of transcription factors. Recruitment of NPR1 to a heterologous promoter by way of the GAL4 DB domain is able to activate expression of a reporter gene in planta, but only in response to SA treatment (Rochon et al., 2006). Based on these observations, it would appear that the ability of NPR1 to function as a coactivator in planta is controlled by its recruitment to an appropriate cis-regulatory element by way of a DNA-binding entity and the SA-dependent stimulation of NPR1 coactivator activity.

The requirement for SA to awaken the coactivator capacity of NPR1 was first indicated by the fact that transgenic lines overexpressing NPR1 do not demonstrate constitutive PR-1 expression (Cao et al., 1998). These over-expressing lines, despite the abundance of NPR1, still require SA treatment to

90 P. Boyle et al.

effect PR-1 activation (Cao et al., 1998). The agent which directly delivers the SA-dependent signal to the latent NPR1 and the modification or switch it produces in the coactivator have not yet been identified. These data suggest that NPR1 could serve as a sensor in the SA signalling pathway, creating a situation in which, upon perception of SA by the cell, a signal is transduced that results in the stimulation of NPR1, rendering it accessible and competent for recruitment to the repressor TGA2. This cascade of events culminates in the formation of the TGA2–NPR1 transactivating entity which activates PR-1 transcription. The presence of multiple sensor elements among the transcription factor–cofactor apparatus, which is able to readily manifest dramatic yet highly directed changes in transcriptional output, is an increasingly popular theme in eukaryotic gene regulation paradigms and one that appears to apply to the regulation of PR-1 in Arabidopsis. Our current understanding of PR-1 regulation is summarized and depicted in Fig. 4.2.

Fig. 4.2. The dual function of TGA2 at the PR-1 promoter. (a) In resting cells TGA2 is recruited to a TGACG motif in the PR-1 promoter in an NPR1-independent manner. TGA2 is required to maintain the PR-1 gene in a repressed state. The coactivator NPR1 is also recruited to the unactivated PR-1 promoter in a TGA2-independent manner at a site yet to be identified (Site X). However, it is not known if this recruitment is mediated by way of an unknown protein (Protein X) or via an uncharacterized DNA binding domain in the NPR1 protein. (b) In salicylic acid (SA)-stimulated cells, TGA2 recruits the coactivator NPR1 and collectively these factors contribute to the formation of an enhanceosome responsible for the activated expression of the PR-1 gene. It is not understood if NPR1 maintains contacts with the agent or site of initial recruitment (Protein X or Site X) following SA stimulation and incorporation into the NPR1–TGA2 enhanceosome. Transactivation by the enhanceosome requires the oxidation of cysteines (C) 521 and 529 located in the transactivation domain (TAD) of NPR1 (adapted from Rochon et al., 2006).

Disease Resistance in Arabidopsis: TGA2 and NPR1 91

Acknowledgements

We thank Ms Jee Yan Chu for editorial assistance. Research in our labs is supported by the NRC PBI core funding (P.R.F.), the National Science and Engineering Research Council (NSERC) discovery grant programme (C.D., P.R.F.), the Canada Foundation for Innovation (C.D.), the Ontario Innovation Trust (C.D.) and the NSERC graduate scholarship programme (P.B.).

References

Cao, H., Bowling, S.A., Gordon, A.S. and Dong, X. (1994) Characterizaton of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance. The Plant Cell 6, 1583–1592.

Cao, H., Li, X. and Dong, X. (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance.

Proceedings of the National Academy of Sciences, USA 95, 6531–6536.

Chang, L.S., Shi, Y. and Shenk, T. (1989) Adeno-associated virus P5 promoter contains an adenovirus E1A-inducible element and a binding site for the major late transcription factor. Journal of Virology 63, 3479–3488.

Conaway, R.C., Sato, S., Tomomori-Sato, C., Yao, T. and Conaway, J.W. (2005) The mammalian Mediator complex and its role in transcriptional regulation. Trends in Biochemical Sciences 30, 250–255.

Delaney, T.P., Friedrich, L. and Ryals, J.A. (1995) Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance. Proceedings of the National Academy of Sciences, USA 92, 6602–6606.

Després, C., DeLong, C., Glaze, S., Liu, E. and Fobert, P.R. (2000) The Arabidopsis NPR1/

NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors. The Plant Cell 12, 279–290.

Fan, W. and Dong, X. (2002) In vivo interaction between NPR1 and transcription factor TGA2 leads to salicylic acid-mediated gene activation in Arabidopsis. The Plant Cell 14, 1377–

1389.

Garcia-Bassets, I., Kwon, Y.S., Telese, F., Prefontaine, G.G., Hutt, K.R., Cheng, C.S., Ju, B.G., Ohgi, K.A., Wang, J., Escoubet-Lozach, L., Rose, D.W., Glass, C.K., Fu, X.D. and Rosenfeld, M.G. (2007) Histone methylation-dependent mechanisms impose ligand dependency for gene activation by nuclear receptors. Cell 128, 505–518.

Gaudin, V., Libault, M., Pouteau, S., Juul, T., Zhao, G., Lefebvre, D. and Grandjean, O. (2001) Mutations in LIKE HETEROCHROMATIN PROTEIN 1 affect flowering time and plant architecture in Arabidopsis. Development 128, 4847–4858.

Gordon, S., Akopyan, G., Garban, H. and Bonavida, B. (2006) Transcription factor YY1:

structure, function, and therapeutic implications in cancer biology. Oncogene 25, 1125–

1142.

Heintzman, N.D. and Ren, B. (2007) The gateway to transcription: identifying, characterizing and understanding promoters in the eukaryotic genome. Cellular and Molecular Life Sciences 64, 386–400.

Hochheimer, A. and Tjian, R. (2003) Diversified transcription initiation complexes expand promoter selectivity and tissue-specific gene expression. Genes & Development 17, 1309–1320.

Hsieh, T.F. and Fischer, R.L. (2005) Biology of chromatin dynamics. Annual Review of Plant Biology 56, 327–351.

92 P. Boyle et al.

Kang, H.G. and Klessig, D.F. (2005) Salicylic acid-inducible Arabidopsis CK2-like activity phosphorylates TGA2. Plant Molecular Biology 57, 541–557.

Kikuchi, A., Kishida, S. and Yamamoto, H. (2006) Regulation of Wnt signaling by protein–

protein interaction and post-translational modifications. Experimental and Molecular Medicine 38, 1–10.

Latchman, D.S. (2001) Transcription factors: bound to activate or repress. Trends in Biochemical Sciences 26, 211–213.

Lebel, E., Heifetz, P., Thorne, L., Uknes, S., Ryals, J. and Ward, E. (1998) Functional analysis of regulatory sequences controlling PR-1 gene expression in Arabidopsis. The Plant Journal 16, 223–233.

Lee, J., He, K., Stolc, V., Lee, H., Figueroa, P., Gao, Y., Tongprasit, W., Zhao, H., Lee, I. and Deng, X.W. (2007) Analysis of transcription factor HY5 genomic binding sites revealed its hierarchical role in light regulation of development. The Plant Cell 19, 731–749.

Leung, T.H., Hoffmann, A. and Baltimore, D. (2004) One nucleotide in a kappaB site can determine cofactor specificity for NF-kappaB dimers. Cell 118, 453–464.

Li, B., Carey, M. and Workman, J.L. (2007) The role of chromatin during transcription. Cell 128, 707–719.

Ma, J. (2005) Crossing the line between activation and repression. Trends in Genetics 21, 54–59.

Martinez, C.A. and Arnosti, D.N. (2008) Spreading of a corepressor linked to action of long-range repressor hairy. Molecular and Cellular Biology 28, 2792–2802.

Mo, X., Kowenz-Leutz, E., Xu, H. and Leutz, A. (2004) Ras induces mediator complex exchange on C/EBP beta. Molecular Cell 13, 241–250.

Mur, L.A., Kenton, P., Lloyd, A.J., Ougham, H. and Prats, E. (2008) The hypersensitive response; the centenary is upon us but how much do we know? Journal of Experimental Botany 59, 501–520.

Mutskov, V. and Felsenfeld, G. (2004) Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9. EMBO Journal 23, 138–149.

Natoli, G. (2004) Little things that count in transcriptional regulation. Cell 118, 406–408.

Naumann, K., Fischer, A., Hofmann, I., Krauss, V., Phalke, S., Irmler, K., Hause, G., Aurich, A.C., Dorn, R., Jenuwein, T. and Reuter, G. (2005) Pivotal role of AtSUVH2 in heterochromatic histone methylation and gene silencing in Arabidopsis. EMBO Journal 24, 1418–1429.

Oostendorp, M., Kunz, W., Dietrich, B. and Staub, T. (2001) Induced disease resistance in plants by chemicals. European Journal of Plant Pathology 107, 19–28.

Pedley, K.F. and Martin, G.B. (2005) Role of mitogen-activated protein kinases in plant immunity. Current Opinion in Plant Biology 8, 541–547.

Pieterse, C.M. and Van Loon, L. (2004) NPR1: the spider in the web of induced resistance signaling pathways. Current Opinion in Plant Biology 7, 456–464.

Rando, O.J. and Ahmad, K. (2007) Rules and regulation in the primary structure of chromatin.

Current Opinion in Cell Biology 19, 250–256.

Ren, Y. and Liao, W.S. (2001) Transcription factor AP-2 functions as a repressor that contributes to the liver-specific expression of serum amyloid A1 gene. Journal of Biological Chemistry 276, 17770–17778.

Rochon, A., Boyle, P., Wignes, T., Fobert, P.R. and Després, C. (2006) The coactivator function of Arabidopsis NPR1 requires the core of its BTB/POZ domain and the oxidation of C-terminal cysteines. The Plant Cell 18, 3670–3685.

Roeder, R.G. (2005) Transcriptional regulation and the role of diverse coactivators in animal cells. FEBS Letters 579, 909–915.

Rosenfeld, M.G., Lunyak, V.V. and Glass, C.K. (2006) Sensors and signals: a coactivator/

corepressor/epigenetic code for integrating signal-dependent programs of transcriptional response. Genes & Development 20, 1405–1428.

Disease Resistance in Arabidopsis: TGA2 and NPR1 93

Sauer, F. and Jackle, H. (1993) Dimerization and the control of transcription by Kruppel.

Nature 364, 454–457.

Sauer, F., Fondell, J.D., Ohkuma, Y., Roeder, R.G. and Jackle, H. (1995) Control of transcription by Kruppel through interactions with TFIIB and TFIIE beta. Nature 375, 162–

164.

Scully, K.M., Jacobson, E.M., Jepsen, K., Lunyak, V., Viadiu, H., Carriere, C., Rose, D.W., Hooshmand, F., Aggarwal, A.K. and Rosenfeld, M.G. (2000) Allosteric effects of Pit-1 DNA sites on long-term repression in cell type specification. Science 290, 1127–1131.

Stancheva, I. (2005) Caught in conspiracy: cooperation between DNA methylation and histone H3K9 methylation in the establishment and maintenance of heterochromatin.

Biochemistry and Cell Biology 83, 385–395.

Struhl, K. (1999) Fundamentally different logic of gene regulation in eukaryotes and prokaryotes. Cell 98, 1–4.

Taatjes, D.J., Marr, M.T. and Tjian, R. (2004) Regulatory diversity among metazoan co-activator complexes. Nature Reviews, Molecular Cell Biology 5, 403–410.

Thomas, M.J. and Seto, E. (1999) Unlocking the mechanisms of transcription factor YY1: are chromatin modifying enzymes the key? Gene 236, 197–208.

Valin, A. and Gill, G. (2007) Regulation of the dual-function transcription factor Sp3 by SUMO.

Biochemical Society Transactions 35, 1393–1396.

Verger, A., Perdomo, J. and Crossley, M. (2003) Modification with SUMO. A role in transcriptional regulation. EMBO Reports 4, 137–142.

Yan, B., Raben, N. and Plotz, P.H. (2002) Hes-1, a known transcriptional repressor, acts as a transcriptional activator for the human acid alpha-glucosidase gene in human fibroblast cells. Biochemical and Biophysical Research Communications 291, 582–587.

Zhang, Y., Tessaro, M.J., Lassner, M. and Li, X. (2003) Knockout analysis of Arabidopsis transcription factors TGA2, TGA5, and TGA6 reveals their redundant and essential roles in systemic acquired resistance. The Plant Cell 15, 2647–2653.

94 © CAB International 2009. Molecular Plant–Microbe Interactions (eds Bouarab et al.)

5 Disease Resistance Genes: Form