Signal Transduction in Systemic Acquired Resistance

Specific interactions between DNA binding proteins and their cognate polynucleotide sequences have been widely regarded as fundamental to transcriptional regulation ever since Jacob and Monod described their model of the lac operon approximately forty years ago. The model not only posited the existence of promoter sequences upon which RNA polymerase positions itself to initiate transcription, but also described accessory proteins that could either interfere or facilitate this process. An understanding of transcriptional regulation thus appeared to be primarily a matter of identifying for each gene (or gene cluster) those repressors, activators, and polynucleotide sequences that interact specifically to allow RNA polymerase to do its job.

It has indeed been possible to tabulate the promoter sequences—or canonical “elements” within promoter regions—that are directly recognized by the prokaryotic RNA polymerase. Canonical elements (e.g., TATA, CAAT, and GC boxes) can also be found upstream of the transcription initiation sites of mammalian genes. It is apparent, however, that eukaryotic transcription is often sensitive to additional elements, functioning either as enhancers or silencers, that can occur at distances of up to a few kilobases away from the transcription initiation site. Proteins that interact with such DNA elements, regardless of whether these reside within “promoter” regions or more distal to the site of transcription initiation, have also proven crucial to the recognition of eukaryotic gene elements by the transcriptional machinery.

Hundreds of protein sequences that represent either established or putative eukaryotic transcription factors now exist in the public databases, and scores of such protein sequences pertain to plant systems. Some of the compiled transcription factors (e.g., TATA box binding proteins) are basic to the ability of the eukaryotic RNA polymerases to form “preinitiation” complexes with promoter regions, and the molecular functionality of many such transcription factors relies on protein–protein interactions as well as direct association with promoter sequences. (For an investigation of specific protein–protein interactions in basal, as well as activated transcription, see Pan et al. [2000] in last month's issue.) Some transcription factors, on the other hand, may be involved in the specific activation of a limited number of genes. Indeed, it is this latter group of factors—those that recognize elements associated with specific promoters—that ultimately regulate transcription in response to signal transduction pathways.

In this issue of THE PLANT CELL, on pages 279–290, Després et al. explore the possible mechanisms by which pathways of signal transduction specific to plants may be effected. The transduction pathway at the center of their study is systemic acquired resistance (SAR), a broad state of immu-nity that arises in response to certain pathogen-induced necrotic lesions (for a review, see Ryals et al., 1996). Although the nature of the transduced signal that initiates SAR is not clear, salicylic acid is indispensable in the relay of the initial signal to the cell nucleus, where specific “SAR genes” are induced. One of these SAR genes is PR-1 (for pathogen-related), the transcription of which can be taken experimentally to represent the molecular culmination of SAR.

The upstream regulatory region of the PR-1 gene has been characterized in some detail (Lebel et al., 1998). Two positive regulatory elements within the region, both of which are required for induction of PR-1 by salicylic acid, manifest consensus sequences for recognition by transcription factors. The first of these elements corresponds to the cognate sequence of the bZIP proteins, a class of transcription factors common to fungi, plants, and animals. bZIP factors are active as dimers, each protomer of which is defined by two domains: one is a basic domain that interacts with specific DNA sequences, and the other—characterized by a leucine zipper—is required for dimerization.

One category of plant bZIP factor that deserves particular mention is the family of TGA proteins; in Arabidopsis, six distinct genes have been heretofore allocated to the TGA family (see Xiang et al., 1997). An ortholog of TGA1 was first recognized in tobacco for its ability to bind with high specificity to the pentanucleotide element (TGACG) within the 35S promoter of the cauliflower mosaic virus (Katagiri et al., 1989). Specific protein–DNA interactions that involve this consensus sequence are typical of all the TGA proteins. Significantly, this same pentanucleotide occurs within the first of the two elements that are necessary for salicylic acid induction of the PR-1 gene (Lebel et al., 1998).

The second of the two elements within the PR-1 promoter appears to correlate not to the specificity of a TGA protein, but rather to the recognition element of NF-κB, a vertebrate transcription factor of the Rel family (Bauerle and Baltimore, 1996). NF-κB has been extensively studied because it functions within various contexts of mammalian signal transduction pathways that lead to the transcription of genes involved in apoptosis, cell growth and differentiation, and immune functions. In addition, NF-κB represents an important model transcription factor because its molecular relationship to higher orders of regulatory control in transduction pathways has been elucidated. Specifically, the ability of NF-κB to activate transcription is controlled in that it remains sequestered in the cytoplasm by an inhibitory factor, IκB, to which it is bound. This inhibitory function, in turn, is regulated by a specific kinase that phosphorylates IκB so as to tag it for ubiquitin-mediated degradation, thereby freeing NF-κB to enter the nucleus and activate the transcription of its target genes (see May and Ghosh, 1999).

It is not yet clear whether SAR involves a transduction pathway analogous to that established for NF-κB in animal systems, and a set of proteins that could support any full-blown pathway is far from established. Nevertheless, Arabidopsis mutants that either mount a constitutive SAR or fail to mount a response under conditions that normally establish SAR have indicated the involvement of specific gene products. One protein that proves to be crucial to SAR is the product of the NPR1 gene (for NONEXPRESSER OF PR-1; also known as NIM1 [for NONINDUCIBLE IMMUNITY1]; Cao et al., 1994; Delaney et al., 1995).

Recently, Zhang et al. (1999) and Zhou et al. (2000) have probed the molecular role of NPR1, which shows no homology to any known transcription factor (Ryals et al., 1997). In particular, these researchers have established that the protein interacts with certain members of the Arabidopsis family of TGA proteins and that at least two of these interact specifically with the PR-1 regulatory region. Mutant NPR1 proteins that fail to support SAR, moreover, were similarly proven to be impaired in their ability to interact with the TGA transcription factors.

In the present report of Després et al., the role of NPR1 is somewhat more broadly addressed to the extent that its interaction with each of the six known TGA products is appraised. Specifically, two of the family members manifestly fail to interact with NPR1 according to a yeast two-hybrid assay. (In vitro analysis of representative members of the family confirms the ability or inability of the factors to interact with NPR1.) In addition, the authors have found a novel TGA factor that interacts with NPR1, thereby expanding the number of TGA representatives in Arabidopsis to seven.

Beyond establishing the varied ability of the entire TGA family to interact with NPR1, Després et al. investigate the possible means by which such interactions may influence gene regulation. Based upon electrophoretic mobility shift assays, the authors reveal that NPR1 can interact with TGA species so as to stimulate their association with cognate oligonucleotide elements. It is not clear whether NPR1 persists within the complexes of TGA factor and DNA. The structural basis for the interaction of the TGA proteins with NPR1 has nevertheless been analyzed by Zhou et al. (2000). Interestingly, their studies of the TGA family suggest that the N termini, where the family members tend to diverge from one another, may influence the varied affinities of the TGA proteins for NPR1.

Clearly, our understanding of SAR remains somewhat cursory. Nevertheless, by establishing NPR1 as a modulator of the interaction between transcription factor and cognate element, Deprés et al. underscore SAR as a bona fide signal transduction pathway that culminates in a downstream series of interactions that influence the molecular recognition of specific gene elements. Given that TGA factors as a single family function to recognize a common regulatory element, the elegance of such modulating activities becomes evident. It is additionally clear that NPR1 function must also be modulated, inasmuch as the protein is also essential in ISR (for induced systemic resistance), a transduction pathway of disease resistance that is distinct from SAR. Just how the function of NPR1 is itself orchestrated at higher levels of cellular control will be an important step in elucidating the mechanisms that underlie both SAR and ISR.