Plant Physiol. (1999) 119: 1243-1250

Fusicoccin, 14-3-3 Proteins, and Defense Responses in Tomato Plants¹

Michael R. Roberts^* and Dianna J. Bowles

The Plant Laboratory, Department of Biology, University of York, P.O. Box 373, York YO10 5YW, United Kingdom

	ABSTRACT

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Fusicoccin (FC) is a fungal toxin that activates the plant plasma membrane H⁺-ATPase by binding with 14-3-3 proteins, causing membrane hyperpolarization. Here we report on the effect of FC on a gene-for-gene pathogen-resistance response and show that FC application induces the expression of several genes involved in plant responses to pathogens. Ten members of the FC-binding 14-3-3 protein gene family were isolated from tomato (Lycopersicon esculentum) to characterize their role in defense responses. Sequence analysis is suggestive of common biochemical functions for these tomato 14-3-3 proteins, but their genes showed different expression patterns in leaves after challenges. Different specific subsets of 14-3-3 genes were induced after treatment with FC and during a gene-for-gene resistance response. Possible roles for the H⁺-ATPase and 14-3-3 proteins in responses to pathogens are discussed.

	INTRODUCTION

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The plant plasma membrane H⁺-ATPase governs the electrochemical gradient across the plasma membrane, which is essential for the control of ion transport and cytoplasmic pH (for review, see Palmgren, 1998). The activity of the H⁺-ATPase is deregulated by the fungal toxin FC, resulting in membrane hyperpolarization and alteration of ionic gradients. This affects a number of plant processes, including cell expansion, seed germination, stomatal behavior, and nutrient uptake (for review, see Marrè, 1979). Changes in membrane potential are also associated with the initiation of a number of other signal transduction pathways (for review, see Ward et al., 1995), in particular those involved in pathogen and in stress responses. The first evidence that FC could affect defense signaling was the inhibition of wound-responsive gene expression in tomato (Lycopersicon esculentum) (Doherty and Bowles, 1990; O'Donnell, 1994). Subsequently, FC was shown to inhibit systemin-induced depolarization of tomato leaf plasma membranes (Moyen and Johannes, 1996), oligogalacturonic acid-induced expression of Phe ammonia lyase (Messiaen and van Cutsem, 1994), and, more recently, elicitation of active oxygen species in cryptogein-treated tobacco cells (Simon-Plas et al., 1997).

FC binds to a single "receptor" in higher plants, and polypeptides isolated as FC-binding proteins from three different plant species have been identified as products of the 14-3-3 gene family (Kourthout and de Boer, 1994; Marra et al., 1994; Oecking et al., 1994). It has now been demonstrated that a functional FC-binding site is formed from a complex between the C-terminal regulatory domain of the H⁺-ATPase and 14-3-3 proteins and that both proteins are required for FC binding (Baunsgaard et al., 1998). However, since another protein-protein complex involving 14-3-3s and the enzyme NR was shown to be disrupted by FC in vitro (Moorhead et al., 1996), the precise mode of interaction between FC and 14-3-3 proteins remains to be established.

14-3-3 proteins are a family of regulatory proteins that have attracted much attention in recent years because of the identification of interactions between various mammalian 14-3-3 isoforms and proteins involved in signal transduction, particularly protein kinases and phosphatases (for review, see Aitken, 1996). Biochemical and structural analyses of 14-3-3 proteins have shown that they occur as homo- and heterodimers in vitro and in vivo, leading to the suggestion that 14-3-3 dimers may mediate interactions between pairs of associated proteins (Jones et al., 1995). It has now been shown that 14-3-3 proteins bind to phosphorylated Ser residues present within one of a small number of consensus sequences found in many of the proteins with which they interact (Muslin et al., 1996; Yaffe et al., 1997; Ku et al., 1998).

A number of 14-3-3 genes have been identified in plants, and functional roles for their products, in addition to their interactions with the H⁺-ATPase, are beginning to be identified. For example, 14-3-3 proteins from Arabidopsis and maize were originally found as part of a transcription factor complex (de Vetten et al., 1992; Lu et al., 1992). It is now known that 14-3-3 proteins complex with the maize transcription factors EmBP1 and VP1 and may function as adapter molecules to establish a complex between the two factors (Schultz et al., 1998). A different role for 14-3-3 proteins is their involvement in the regulation of NR (Bachmann et al., 1996; Moorhead et al., 1996). NR is regulated by phosphorylation, and its activity is inhibited when 14-3-3 proteins are bound specifically to the phosphorylated form of the protein.

We are interested in understanding the regulation of signal transduction pathways leading to responses to pathogens and stress. The aim of this study was to investigate the effect of FC on PR signaling and to identify tomato 14-3-3 gene products that may be involved. We describe the 14-3-3 gene family of tomato and identify the responses of individual members to FC and PR signals.

Accession numbers for the sequences reported in this article are: X95900 (TFT1), X95901 (TFT2), X95902 (TFT3), X95903 (TFT5), X95904 (TFT6), X95905 (TFT7), X98864 (TFT8), X98865 (TFT9), and X98866 (TFT10).

	MATERIALS AND METHODS

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Plant Material

14-3-3 cDNA Cloning by PCR

Analysis of DNA and Protein Sequence and Structure

DNA and RNA Analysis

	RESULTS

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The Effect of FC on PR Gene Expression

View larger version (36K): [in this window] [in a new window]   Figure 1. Effects of FC on PR gene expression. A, PR gene expression is induced by FC, as shown by RNA gel blots showing expression of PR1 (accession no. M69248), acidic PR glucanase (PR2A, accession no. M80604), acidic PR chitinase (PR3A, accession no. Z15141), basic PR chitinase (PR3B, accession no. Z15140), and ACC oxidase (ACO, accession no. A35021) in tomato leaves pretreated with either 1 µM FC or water for 30 min. Hybridization to a 28S rRNA probe is shown as a loading control. The data are representative of at least three independent experiments. B, Synergistic effects of FC and avirulence elicitor on PR gene expression, as shown by RNA gel blots showing expression of acidic PR chitinase (PR3A), basic PR chitinase (PR3B), and ACO in tomato cotyledons 24 h after injection with combinations of 1 µM FC and intercellular fluid extracts containing (Avr9+) or lacking (Avr9) the Avr9 elicitor.

To investigate the effects of FC on a resistance (R) gene response, we used the well-characterized response induced by the Cladosporium fulvum race-specific elicitor Avr9 in tomato plants carrying the Cf9 R gene (for review, see Hammond-Kosack and Jones, 1995). Co-infiltration of 1 µM FC with Avr9-containing intercellular fluids into cotyledons of Cf9 seedlings produced no visible effect on hypersensitive lesion formation. However, at the level of gene expression, a greater accumulation of the acidic and basic chitinases was observed in the co-infiltrated leaves (Fig. 1B). The co-infiltration also caused a severe downward curvature of the cotyledon petioles that was absent after treatment with Avr9 or FC alone. This resembled a typical epinastic response, which suggests increased ethylene production in the co-infiltrated cotyledons.

Tomato Contains at Least 10 Members of the FC-Binding 14-3-3 Protein Family

Alignment of the nucleotide sequences of the 3-RACE products enabled a second degenerate oligonucleotide primer to be designed for 5-RACE amplifications. The overlap between the 3- and 5-RACE cDNA clones was used to match corresponding products and to construct full-length cDNA sequences. In addition to the original six cDNAs whose sequences were completed in this way, the 5 regions of four other 14-3-3 cDNAs were identified. Each of the 10 classes of cDNA clones is derived from a unique gene. We have designated these 10 genes TFT1 to TFT10 (Tomato Fourteen-Three-three). The genes described here as TFT3 and TFT4 correspond to those described by Laughner et al. (1995) as Le GF14 T3 and Le GF14 T4, respectively (accession nos. L29151 and L29150), except that the TFT3 sequence is full length.

Table I presents a summary indicating the level of amino acid identity between the deduced translation products of the TFT genes and, for comparison, between the TFT sequences and human 14-3-3. A dendrogram derived from an alignment of the tomato 14-3-3 sequences, human 14-3-3 and 14-3-3, and the 10 Arabidopsis 14-3-3 gene family members shows that the plant 14-3-3 proteins fall into two major groups. Each group is distinct from 14-3-3, but one includes 14-3-3 (Fig. 2). These groups correspond to those described by Wu et al. (1997). The dendrogram also suggests that there is not an equivalent tomato 14-3-3 protein for each Arabidopsis 14-3-3 protein.

View this table: [in this window] [in a new window]   Table I. Amino acid identity between TFT genes and human 14-3-3 Percent identities were calculated using the Gap program (Genetics Computer Group). Only the N-terminal region covered by all tomato sequences was considered in the comparison.

View larger version (20K): [in this window] [in a new window]   Figure 2. Relationship among tomato, Arabidopsis, and human 14-3-3 sequences. Dendrogram was produced by the GCG program PileUp and represents amino acid sequence similarity between tomato and Arabidopsis (gf protein) 14-3-3 proteins and human 14-3-3 (Swissprot 143T_human) and 14-3-3 (Swissprot 143E_human). Only the N-terminal region covered by all tomato sequences was considered in the comparisons.

Predicted Structural Features of Tomato 14-3-3s

View larger version (39K): [in this window] [in a new window]   Figure 3. Similarity between tomato 14-3-3 and human 14-3-3. A, Conserved regions in 14-3-3 proteins correspond to -helices, as shown by amino acid sequence alignment between a consensus sequence derived from the six full-length tomato cDNAs and human 14-3-3. Only amino acids conserved between all six tomato sequences are shown (top line); identity between tomato and 14-3-3 residues is indicated by a bar. Gaps (periods) were introduced to optimize the alignment. The regions that form the nine -helices in the 14-3-3 crystal structure are indicated by a line below the sequence. B, Sequence homology is mainly within the inner face of 14-3-3 proteins, as shown by a space-filling model of the 14-3-3 protein crystal structure colored by homology with the tomato consensus shown in A. Identical amino acids are shown in blue, and nonidentical residues are shown in yellow. The upper inner face of the molecule, which corresponds to the C-terminal domain, shows high sequence conservation; whereas the lower inner face, corresponding to the N-terminal dimerization domain, and the whole outer face are poorly conserved.

14-3-3 Genes Exhibit Differential Regulation in Challenged Leaves

View larger version (50K): [in this window] [in a new window]   Figure 4. Southern analysis of the tomato 14-3-3 gene family. 5 Probes from the 10 tomato 14-3-3 cDNAs were hybridized at high stringency to identical filters containing genomic DNA digested with EcoRI+BamHI (left lanes) and HindIII (right lanes). The positions of molecular size standards are indicated to the left of the figure.

We first examined expression of the TFT genes after treatment of plants with FC. Solutions of 1 µM FC were applied to the cut stems of plants for 30 min for uptake via the transpiration stream. Figure 5 shows the expression patterns of the TFT genes in leaves over a time course from a representative experiment. Steady-state transcript levels of most of the genes appear relatively unchanged, but significant up-regulation of four genes, TFT4, TFT8, TFT9, and TFT10, was observed repeatedly. The time course of induction of these genes was similar to the induction of PR genes by FC. We then examined the expression of 14-3-3 genes during the Cf9-Avr9 R gene response and found that most members of the gene family were unresponsive, with no significant change in steady-state transcript level after injection of Avr9 intercellular fluid into Cf9 leaves.

View larger version (50K): [in this window] [in a new window]   Figure 5. Tomato 14-3-3s are differentially expressed in response to FC. Time points showing the mRNA levels in tomato leaves of the 10 tomato 14-3-3 genes through a time course after a 30-min treatment with 1 µM FC. RNA from leaves of healthy, untreated plants and from leaves of plants incubated in water for 8 h are included as references. Hybridization to a 28S rRNA probe is shown as a loading control. The data are representative of at least three independent experiments.

The results of using TFT5 and TFT9 probes are shown in Figure 6 as examples of this lack of response. In contrast, the expression of three genes, TFT1, TFT4, and TFT6, was induced, and the induction was specific to the R gene response since no changes were observed on injection of intercellular fluid into Cf0 plants, which do not carry the corresponding R gene. Different time courses of induction were observed for each of these three genes. TFT4 already showed maximum steady-state mRNA levels by the first time point sampled (4 h), and TFT6 showed a transient accumulation of transcripts to a maximum at 16 h, whereas TFT1 steady-state mRNA levels showed a gradual increase over the entire time course of 48 h, corresponding closely to the time course of induction of the PR1 gene used as a control for a positive response (Fig. 6).

View larger version (80K): [in this window] [in a new window]   Figure 6. Tomato 14-3-3s are differentially expressed during a resistance response. Time points showing the mRNA levels in Cf9 and Cf0 tomato leaves of selected tomato 14-3-3 genes through a time course after infiltration of Avr9-containing intercellular fluid. Hybridization to a 28S rRNA probe is shown as a loading control. The data are representative of two independent experiments.

	DISCUSSION

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FC has often been used as an experimental tool to investigate the role of plasma membrane depolarization in signaling events leading to plant defense responses. Through its deregulation of the H⁺-ATPase, FC treatment hyperpolarizes the plasma membrane, and this can inhibit downstream responses dependent on membrane depolarization.

It has been shown in tomato that leaf damage or application of oligogalacturonic acids or of the systemin peptide leads to instantaneous membrane depolarization (Thain et al., 1990; Moyen and Johannes, 1996). Pretreatment with FC blocks this depolarization and the effects of wounding and elicitors on the expression of wound-responsive marker genes, such as those encoding proteinase inhibitors (Doherty and Bowles, 1990; Messiaen and van Cutsem, 1994; O'Donnell, 1994). Similarly, the induction of reactive oxygen species by cryptogein, thought in tobacco to act through suppression of H⁺-ATPase activity, is inhibited by pretreatment with FC (Simon-Plas et al., 1997). In contrast, in this study we show that when FC is co-infiltrated with Avr9 elicitor into tomato plants carrying the Cf9 R gene the compound leads to a synergistic effect on PR gene expression. Since our control infiltrations included intercellular fluid preparations lacking Avr9 peptide, this synergism was due to a specific interaction between the Avr9- and FC-induced signals, not some other component in the intercellular fluid.

Our data are consistent with previous studies reporting a R-gene-dependent activation of the plasma membrane H⁺-ATPase in response to C. fulvum Avr5 avirulence factor (Vera-Estrella et al., 1994; Xing et al., 1996). It is possible that increased stomatal opening and water loss are part of the normal process of localized PR gene induction in tissues undergoing lesion formation and that FC exacerbates these effects. The importance of water status is highlighted by the observation that high humidity suppresses many aspects of the Cf2- and Cf9-related resistance responses in tomato (Hammond-Kossack et al., 1996). It is also known that a wide range of plant pathogens produce compounds that directly affect H⁺-ATPase activity (for review, see Knogge, 1996), e.g. supprescin B synthesized by virulent races of Mycosphaerella pinodes, which inhibits H⁺-ATPase activity and renders plants more susceptible to avirulent races of the pathogen.

We found that application of FC alone induces PR gene expression in tomato plants. As yet we do not know whether this is a direct or an indirect consequence of the compound's effect on the H⁺-ATPase or if there may be some other biochemical target for FC. The effect of FC on ethylene emission from suspension-cultured cells was shown previously and was found to be dependent on the activation of the H⁺-ATPase (Malerba et al., 1995; Malerba and Bianchetti, 1996). FC also induces ethylene when applied to tomato plants (O'Donnell, 1994), and although ethylene is known to induce basic PR gene expression in tobacco (Ohme-Takagi and Shinshi, 1995), in the present study preferential induction of the acidic rather than the basic PR protein gene was observed.

At the molecular level only a single class of FC-binding proteins is detectable, which are now known to be products of the 14-3-3 gene family. Initially, it was thought that the 14-3-3 proteins bound FC directly, but recently it has been shown that FC binds only to a protein-protein complex composed of a 14-3-3 protein and the C terminus of the H⁺-ATPase (Baunsgaard et al., 1998). The exclusivity of this target has been questioned by Moorhead et al. (1996), with data showing that FC also affects complex formation between 14-3-3s and NR. However, the effect on NR was demonstrated only in vitro and at FC concentrations much higher than those required for its biological activity.

We have found that tomato plants possess at least 10 14-3-3 genes and that these exhibit differential patterns of expression in the leaf, responding differently to FC and elicitor treatments. These data extend investigations of 14-3-3 gene expression in Arabidopsis and tomato that analyzed tissue-specific expression (Laughner et al., 1995; Daugherty et al., 1996). The question as to whether individual 14-3-3 proteins exhibit functional specificity is currently a matter for debate. In support of this possibility, some reports indicate a preferential association between particular 14-3-3 proteins and target proteins (e.g. human A20 and 14-3-3 [Vincenz and Dixit, 1996]), whereas Drosophila melanogaster 14-3-3 mutants exhibit distinct developmental phenotypes, including lethality for 14-3-3 (Kockel et al., 1997).

At the protein level there are structural data and a range of biochemical analyses in vitro to argue against a specific role for each 14-3-3 gene product. For example, Yaffe et al. (1997) demonstrated that all of the six human and two yeast 14-3-3 recombinant gene products that they analyzed showed very similar binding characteristics in vitro to phosphopeptide sequences. The major sites of contact with these interacting phosphopeptides are conserved residues found in all mammalian and yeast 14-3-3s and located within the groove formed by a 14-3-3 dimer, as visualized in the crystal structure of the human  protein (Xiao et al., 1995). Any specificity of function of the different 14-3-3 gene products in the cell was suggested to arise via interactions between target proteins and the less-conserved residues on the external face of the dimer (Yaffe et al., 1997). Comparing the tomato 14-3-3 sequences with those of other eukaryotic 14-3-3 genes shows greatest structural conservation within the groove. Thus, it is highly probable that all plant 14-3-3 gene products have the ability to bind the same phosphopeptides in vitro, and this lack of specificity between plant 14-3-3 recombinant proteins and a range of targets has been demonstrated in biochemical studies (Lu et al., 1994; Moorhead et al., 1996; van Heusden et al., 1996; Baunsgaard et al., 1998).

In the plant, functional specificity could be endowed by the cell-specific, inducible, or developmental regulation of 14-3-3 gene expression, such that only a subset of 14-3-3 proteins is present in a cell at any one time. In this context, transcriptional control of 14-3-3 gene expression has recently been shown to be important in events mediated at the protein level. Thus, transcription of the human 14-3-3 form was directed by an activated p53 tumor-suppressor protein in response to DNA damage, and the newly synthesized 14-3-3 protein bound to and sequestered Cdc25c, leading to cell-cycle arrest (Hermeking et al., 1997).

It is possible that the responses of the tomato 14-3-3 genes to different stimuli reflect causal roles of their products in the plant. To address this issue we are in the process of producing transgenic lines in which individual 14-3-3 genes are eliminated by antisense mRNA expression. Studies are also in progress to identify targets of 14-3-3 proteins in tomato leaves and to define their interactions during defense responses.

	FOOTNOTES

   Received October 26, 1998; accepted January 14, 1999.

	ABBREVIATIONS

Abbreviations: FC, fusicoccin. NR, nitrate reductase. PR, pathogenesis-related. RACE, rapid amplification of cDNA ends.

	ACKNOWLEDGMENTS

The authors thank Dr. J. van Kan and Professor P.J.G.M. de Witt (Wageningen Agricultural University, The Netherlands) for the generous gift of the tomato PR gene cDNAs and Robert Darby (University of Wales, Aberystwth, UK) for the rRNA cDNA. The coordinates for the 14-3-3 protein structure were kindly made available by Dr. Steve Gamblin (National Institute for Medical Research, London). Tomato Avr9-containing intercellular fluid was generously provided by Dr. Jonathan Jones (Sainsbury Laboratory, John Innes Centre for Plant Science Research, Norwich, UK). We also thank the members of the European Community Central Role in Adaptation of Fourteen Three Three Proteins network for their helpful discussions.

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