PBF-2 Is a Novel Single-Stranded DNA Binding Factor Implicated in PR-10a Gene Activation in Potato

Abstract

Elicitor-induced activation of the potato pathogenesis-related gene PR-10a requires a 30-bp promoter sequence termed the ERE (elicitor response element) that is bound by the nuclear factor PBF-2 ( PR-10a binding factor 2). In this study, PBF-2 has been purified to near homogeneity from elicited tubers through a combination of anion-exchange and DNA affinity chromatography. Evidence demonstrates that inactive PBF-2 is stored in the nuclei of fresh tubers and becomes available for binding to the ERE upon elicitation. A protein with an apparent molecular mass of 24 kD (p24) is a DNA binding component of PBF-2. A cDNA encoding p24 has been cloned and encodes a novel protein with a potential transcriptional activation domain that could also act as a single-stranded DNA binding domain. Both PBF-2 and the cDNA-encoded protein bind with high affinity to the single-stranded form of the ERE in a sequence-specific manner. The inverted repeat sequence of the ERE, TGACAnnnnTGTCA, is critical for binding of this factor in vitro and for PR-10a expression in vivo, supporting the role of PBF-2 as a transcriptional regulator. INTRODUCTIONPlants defend themselves against fungal pathogens by a variety of mechanisms, including preexisting physical barriers and inducible defenses (Lamb et al., 1989). Attack by an avirulent strain of pathogen results in a rapid localized necrosis at the site of infection (termed the hypersensitive response), which contributes to pathogen limitation (Keen, 1992). With a few exceptions, deployment of the inducible defenses requires massive gene induction (Lamb et al., 1989). Despite the importance of transcriptional activation during the plant defense response, very little is known about the players involved and the exact mechanisms that lead to defense gene induction.PR (pathogenesis-related) genes are among the best characterized genes induced by pathogens. Heterogeneous in structure and function, PR genes are subdivided into 11 groups (Van Loon et al., 1994). Although the function of certain PR proteins is unknown, some display in vitro antifungal properties (Schlumbaum et al., 1986;Vigers et al., 1991;Ponstein et al., 1994;Niderman et al., 1995). Genes of the PR-10 group are present in numerous dicots (Somssich et al., 1988;Breiteneder et al., 1989;Matton and Brisson, 1989;Walter et al., 1990) and monocots (Warner et al., 1992;Moons et al., 1997). Evidence is accumulating that some PR-10 proteins might possess ribonuclease activity (Moiseyev et al., 1994;Bufe et al., 1996;Swoboda et al., 1996). More recently, structural and sequential homology between the PR-10 proteins and a group of latex proteins has been described (Osmark et al., 1998). Genes of the PR-10 group encode small, primarily acidic intracellular proteins with molecular masses ranging from 15 to 18 kD and have been shown to be transcriptionally regulated (Linthorst, 1991).In only two cases have cis elements and their trans -acting factors been characterized in the promoters of PR-10 genes. These studies revealed that the processes of tr...

Cloning and Biochemical Analysis of the TetrahymenaOrigin Binding Protein TIF1

“…Whereas p24 recognizes a palindromic sequence present on both strands, TIF1 binds to unique sequences in the complementary A-rich and T-rich strands (25). However, both proteins appear to bind to a single DNA substrate molecule in vitro, suggesting that they do not form a bridge within melted duplex DNA (25,30). This was unexpected for TIF1, as the native protein is a homotetramer with at least four potential type I element binding sites.…”

“…The Solanum p24 protein and predicted translation product of the Arabidopsis cDNA share 80% identity, suggesting that they are closely related in function. p24 is an integral component of a multiprotein complex that activates transcription by binding to cis-acting determinants in the PR-10a promoter (30). Two clustered sites of sequence conservation were observed between TIF1 and the two plant proteins, corresponding to 21-and 23-amino acid blocks that span 24% of the mature TIF1 protein.…”

“…3B). Remarkably, one of these proteins, p24 from Solanum tuberosum (potato), is a sequence-specific single-stranded DNA binding factor (30). p24 is an integral part of a multiprotein complex, PBF-2, that induces transcription of the pathogen-resistance gene PR10a, upon exposure to plant pathogens.…”

“…The homologous regions encompass 24% of the TIF1 protein (region 1: 34% identity, 61% similarity to p24; region 2: 24% identity, 81% similarity to p24). TIF1 lacks the polyglutamine tract proposed to play a role in transcriptional activity by p24 (30,37).…”

“…We demonstrate that TIF1 binds to single-stranded type I elements in vivo, as well as to an additional phylogenetically conserved sequence, the pause site element (PSE), that is shown here to be essential. TIF1 contains limited sequence similarity to a sequence-specific single-stranded DNA-binding protein in plants (30), suggesting that these proteins may share structural features for target DNA recognition. Reverse genetic studies argue that Tetrahymena rDNA metabolism (replication initiation, replication elongation, and rRNA transcription) is regulated by multiple PSE and type I element binding proteins, some of which may have evolved overlapping, complementary functions.…”