Repression domains of class II ERF transcriptional repressors share an essential motif for active repression.

We reported previously that three ERF transcription factors, tobacco ERF3 (NtERF3) and Arabidopsis AtERF3 and AtERF4, which are categorized as class II ERFs, are active repressors of transcription. To clarify the roles of these repressors in transcriptional regulation in plants, we attempted to identify the functional domains of the ERF repressor that mediates the repression of transcription. Analysis of the results of a series of deletions revealed that the C-terminal 35 amino acids of NtERF3 are sufficient to confer the capacity for repression of transcription on a heterologous DNA binding domain. This repression domain suppressed the intermolecular activities of other transcriptional activators. In addition, fusion of this repression domain to the VP16 activation domain completely inhibited the transactivation function of VP16. Comparison of amino acid sequences of class II ERF repressors revealed the conservation of the sequence motif (L)/(F)DLN(L)/(F)(x)P. This motif was essential for repression because mutations within the motif eliminated the capacity for repression. We designated this motif the ERF-associated amphiphilic repression (EAR) motif, and we identified this motif in a number of zinc-finger proteins from wheat, Arabidopsis, and petunia plants. These zinc finger proteins functioned as repressors, and their repression domains were identified as regions that contained an EAR motif.

Regulation of ethylene-induced transcription of defense genes.

Ethylene-induced gene expression has been studied in systems in which the biosynthesis of ethylene is stimulated during developmental process such as ripening of fruit, senescence of flower petals, or during pathogen infection. Functional analysis of the promoters of these genes revealed that the ethylene-responsive cis-elements of fruit ripening genes and senescence genes differed from that of defense genes whose expression is induced by ethylene in response to pathogen infection. The ethylene-responsive element identified as the GCC box (AGCCGCC) is commonly found in the promoter region of the ethylene-inducible defense genes. The ethylene responsive element binding factors that interact with the GCC box were demonstrated to be the transcription factors, which respond to extracellular signals to modulate GCC box-mediated gene expression positively or negatively.

Characterization of gene expression of NsERFs, transcription factors of basic PR genes from Nicotiana sylvestris.

Three genes of NsERFs (EREBPs), transcription factors for GCC box of basic PR genes, were isolated from Nicotiana sylvestris. Analyses of transgenic tobacco carrying the NsERF promoter::GUS genes showed that expression of all NsERF genes in leaves was induced by ethylene. Sequence analyses indicated that the 5'-upstream region of NsERF3 gene has the GCC box. In contrast, the promoter regions of NsERF2 and 4 have no GCC box, whereas NsERF2 has a putative EIN3 binding site. Tissue/cell specific expression is also discussed.

Three ethylene-responsive transcription factors in tobacco with distinct transactivation functions.

Ethylene-responsive factors (ERFs) have conserved DNA-binding domains and interact directly with the GCC box in the ethylene-responsive element that is necessary and sufficient for the regulation of transcription by ethylene. ERFs were shown to be localized to nucleus in transient transfection experiments. Transient expression assays using tobacco protoplasts and a heterologous system in yeast were used to examine the transactivation functions of ERFs. ERF2 and ERF4 enhanced the GCC box-mediated transcription of a reporter gene in tobacco protoplasts. When fused to the DNA-binding domain of yeast GAL4, a carboxy-terminal region of ERF2, as well as both amino-terminal and carboxy-terminal regions of ERF4, functioned as a transactivation domain in tobacco protoplasts. The amino-terminal regions of ERF2 and ERF4 functioned as transactivation domains in yeast. In contrast to ERF2 and ERF4, ERF3 reduced the transcription of the reporter gene in tobacco protoplasts, indicating that ERF3 functions as a repressor. Thus, it appears that ERFs exert their regulatory functions in different ways, with ERF2 and ERF4 being activators and ERF3 being a repressor of transcription.

Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression.

Ethylene-responsive element binding factors (ERFs) are members of a novel family of transcription factors that are specific to plants. A highly conserved DNA binding domain known as the ERF domain is the unique feature of this protein family. To characterize in detail this family of transcription factors, we isolated Arabidopsis cDNAs encoding five different ERF proteins (AtERF1 to AtERF5) and analyzed their structure, DNA binding preference, transactivation ability, and mRNA expression profiles. The isolated AtERFs were placed into three classes based on amino acid identity within the ERF domain, although all five displayed GCC box-specific binding activity. AtERF1, AtERF2, and AtERF5 functioned as activators of GCC box-dependent transcription in Arabidopsis leaves. By contrast, AtERF3 and AtERF4 acted as repressors that downregulated not only basal transcription levels of a reporter gene but also the transactivation activity of other transcription factors. The AtERF genes were differentially regulated by ethylene and by abiotic stress conditions, such as wounding, cold, high salinity, or drought, via ETHYLENE-INSENSITIVE2 (EIN2)-dependent or -independent pathways. Cycloheximide, a protein synthesis inhibitor, also induced marked accumulation of AtERF mRNAs. Thus, we conclude that AtERFs are factors that respond to extracellular signals to modulate GCC box-mediated gene expression positively or negatively.

Slow and prolonged activation of the p47 protein kinase during hypersensitive cell death in a culture of tobacco cells

To investigate the involvement of protein kinases in the signaling cascade that leads to hypersensitive cell death, we used a previously established system in which a fungal elicitor, xylanase from Trichoderma viride (TvX), induces a hypersensitive reaction in tobacco (Nicotiana tabacum) cells in culture (line XD6S). The elicitor induced the slow and prolonged activation of a p47 protein kinase, which has the characteristics of a family member of the mitogen-activated protein kinases. An inhibitor of protein kinases, staurosporine, and a blocker of Ca channels, Gd3+ ions, both of which blocked the TvX-induced hypersensitive cell death, inhibited the TvX-induced activation of p47 protein kinase. Moreover, an inhibitor of serine/threonine protein phosphatase alone induced both rapid cell death and the persistent activation of the p47 protein kinase. Thus, the p47 protein kinase might be a component of the signal transduction pathway that leads to hypersensitive cell death, and the regulation of the duration of activation of the p47 protein kinase might be important in determining the destiny of tobacco cells.

Elicitor-responsive, ethylene-independent activation of GCC box-mediated transcription that is regulated by both protein phosphorylation and dephosphorylation in cultured tobacco cells.

In cultured XD6S tobacco cells, xylanase from Trichoderma viride (TvX) induced the expression of a luciferase reporter gene that was under the control of a GCC box, which is an 11 bp sequence (TAAGAGCCGCC) that is found in the 5'-upstream region of pathogen-responsive defence genes that include genes for class I basic chitinase. TvX-induced biosynthesis of ethylene was not required for the TvX-activated transcription. The TvX-induced, GCC box-mediated transcription of the reporter gene was completely blocked not only by staurosporine, an inhibitor of serine/threonine protein kinases, at 1 microM, but also by calyculin A, an inhibitor of protein phosphatases 1 and 2A, at 0.2 microM. It appeared also that protein synthesis de novo was required for the GCC box-mediated transcription of the reporter gene. Accumulation of mRNAs for various ERFs (ethylene-responsive transcription factors), which have been shown to bind specifically to the GCC box, was also induced by TvX prior to increases in the level of mRNA for a class I basic chitinase. In particular, the level of mRNA for EFR2 reached a maximum from 3 to 6 h, whereas levels of mRNAs for ERF3 and ERF4 were highest 0.5 h after the start of treatment of TvX and decreased thereafter. Moreover, induction of accumulation of the mRNA for ERF2 was inhibited by staurosporine and calyculin A. These results suggest that ERF2 might play a major role in TvX-induced, GCC box-mediated transcription of genes and that both protein kinase(s) and protein phosphatase(s) might be involved, as positive regulators, in the signal transduction pathway that leads to expression of ERF2 and subsequent GCC box-mediated transcription of genes.

A tobacco gene encoding a novel basic class II chitinase: a putative ancestor of basic class I and acidic class II chitinase genes.

Various chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains. We have isolated a tobacco gene that encodes a predicted polypeptide consisting of a 20-amino acid N-terminal signal peptide, followed by a 245-amino acid chitinolytic domain. Although the predicted mature protein is basic and shows greater sequence identity to basic class I chitinases (75%) than to acidic class II chitinases (67%), it lacks the N-terminal cysteine-rich domain and the C-terminal vacuolar targeting signal that is diagnostic for class I chitinases. Therefore, this gene appears to encode a novel, basic, class II chitinase, which we have designated NtChia2;B1. Accumulation of Chia2;B1 mRNA was induced in leaves in association with the local-lesion response to tobacco mosaic virus (TMV) infection, and in response to treatment with salicylic acid, but was only slightly induced by treatment with ethephon. Little or no Chia2;B1 mRNA was detected in roots, flowers, and cell-suspension cultures, in which class I chitinase mRNAs accumulate to high concentrations. Sequence comparisons of Chia2;B1 with known tobacco class I and class II chitinase genes suggest that Chia2;B1 might encode an ancestral prototype of the present-day class I and class II isoforms. Possible mechanisms for chitinase gene evolution are discussed.

Transient Activation and Tyrosine Phosphorylation of a Protein Kinase in Tobacco Cells Treated with a Fungal Elicitor.

Suspension-cultured tobacco cells respond to fungal elicitor by activating the transcription of so-called defense genes. This response has been shown to be blocked by staurosporine, an inhibitor of protein kinases, and by Gd3+, which blocks Ca2+ channels. We report here that treating tobacco cells with the elicitor triggers the rapid and transient activation of a 47-kD protein kinase that phosphorylates serine and/or threonine residues of the myelin basic protein (MBP). Staurosporine and Gd3+ inhibited the elicitor-induced activation of the 47-kD MBP kinase, and staurosporine inhibited the activity of the MBP kinase itself. In the presence of either cycloheximide or calyculin A, the elicitor induced sustained activation of the MBP kinase. Immunoblot and immunoprecipitation analysis using a phosphotyrosine-specific antibody showed that tyrosine phosphorylation of the 47-kD MBP kinase was induced in tobacco cells that had been treated with the elicitor. The results suggest that the 47-kD MBP kinase is a component of the pathway for transduction of the elicitor signal in tobacco cells and that the activity of the MBP kinase is regulated by the post-translational phosphorylation of tyrosine residues.

Identification of an ethylene-responsive region in the promoter of a tobacco class I chitinase gene.

The Chn48 gene is a representative of a family of tobacco class I basic chitinase genes, and the expression is induced by the stress hormone ethylene. To investigate the molecular basis for transcriptional regulation by ethylene we have examined the Chn48 promoter to identify cis-elements and trans-acting factors that are involved in the chitinase gene expression. In transgenic tobacco plants, a chimeric gene construct containing a 2 kb Chn48 promoter fused to a beta-glucuronidase reporter gene was induced by ethylene in leaf tissues. Deletion analysis indicated that a positive ethylene-responsive region is located between nucleotides -503 and -358 relative to the transcription initiation site. This 146 bp sequence was found to confer ethylene-responsive reporter gene expression when inserted in either orientation upstream of the heterologous promoter, indicating that the sequence functions as a regulatory enhancer. The ethylene-responsive region contains two copies of a GCC-box (TAAGAGCCGCC), which is conserved in a number of ethylene-responsive defense genes. The sequences within this ethylene-responsive region that are necessary for ethylene-responsive transcription were further localized to the 71 bp sequence between positions -480 and -410 containing two copies of the GCC-box by loss-of-function analysis. Gel mobility-shift experiments showed the presence of leaf nuclear factors that interact with the DNA sequences included in the ethylene-responsive region.

Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element.

We demonstrated that the GCC box, which is an 11-bp sequence (TAAGAGCCGCC) conserved in the 5' upstream region of ethylene-inducible pathogenesis-related protein genes in Nicotiana spp and in some other plants, is the sequence that is essential for ethylene responsiveness when incorporated into a heterologous promoter. Competitive gel retardation assays showed DNA binding activities to be specific to the GCC box sequence in tobacco nuclear extracts. Four different cDNAs encoding DNA binding proteins specific for the GCC box sequence were isolated, and their products were designated ethylene-responsive element binding proteins (EREBPs). The deduced amino acid sequences of EREBPs exhibited no homology with those of known DNA binding proteins or transcription factors; neither did the deduced proteins contain a basic leucine zipper or zinc finger motif. The DNA binding domain was identified within a region of 59 amino acid residues that was common to all four deduced EREBPs. Regions highly homologous to the DNA binding domain of EREBPs were found in proteins deduced from the cDNAs of various plants, suggesting that this domain is evolutionarily conserved in plants. RNA gel blot analysis revealed that accumulation of mRNAs for EREBPs was induced by ethylene, but individual EREBPs exhibited different patterns of expression.

Characterization of a novel cis-acting element that is responsive to a fungal elicitor in the promoter of a tobacco class I chitinase gene.

The expression of tobacco class I chitinase gene is effectively induced by a fungal elicitor in suspension-cultured tobacco cells. To identify cis-acting DNA elements that respond to the elicitor, a series of promoter constructs of the chitinase gene CHN50 fused to beta-glucuronidase gene was introduced into tobacco cultured cells. Promoter deletion analysis of the chitinase gene CHN50 in transgenic tobacco calli indicated that the DNA region between positions -788 and -345 from the start site of transcription is required for inducibility by the elicitor. A gel mobility shift assay revealed that nuclear factor(s) specifically interacted with the DNA region between positions -574 and -476. Moreover, this novel DNA-binding activity was present in nuclear extracts prepared from elicitor-treated cultured cells but not in extracts from untreated cells. Competitive binding assays and methylation interference experiments showed that the nuclear factor(s) bound specifically to a sequence of 22 bp that extended from positions -539 to -518 and contained a direct repeat of GTCAG spaced by three nucleotides. This motif is a candidate for a cis-acting elicitor-responsive element (ElRE) that is involved in the transcription of the class I chitinase gene.

The structure and regulation of homeologous tobacco endochitinase genes of Nicotiana sylvestris and N. tomentosiformis origin.

The fungicidal class I chitinases are believed to be important in the induced defense response of plants. We isolated and partially characterized genomic clones representing two members, CHN14 and CHN50, of the gene subfamily encoding these enzymes in Nicotiana tabacum L. cv. Havana 425. The coding sequences of genes CHN14, CHN50, and CHN48, which was cloned earlier, are identical at 79-95% of the positions. Tobacco is an amphidiploid species derived from ancestors most closely related to the present-day species N. sylvestris and N. tomentosiformis. Southern analysis of genomic DNA, comparison of deduced amino acid sequences, and partial sequencing of the purified enzymes suggest that the gene pairs CHN48/CHN50 and CHN14/CHN14' are homeologues. Gene CHN48, which encodes chitinase A (Mr ca. 34 kDa), and gene CHN14 are derived from N. tomentosiformis; whereas gene CHN50, which encodes chitinase B (Mr ca. 32 kDa), and gene CHN14' are derived from N. sylvestris. Class I chitinases are induced in leaves of plants treated with ethylene or infected with the fungal pathogen Cercospora nicotianae and in cultured cells transferred to medium without added auxin and cytokinin. RNase protection assays show that under these conditions transcripts encoded by the homeologues CHN48 and CHN50 account for greater than 90% of the total chitinase mRNA. The less abundant transcript, CHN48, consistently showed a greater degree of induction than CHN50. Expression of the homeologues CHN14 and CHN14' represented less than 10% of the total chitinase mRNA. They showed a pattern of hormonal regulation similar to CHN48 and CHN50, but transcripts of these genes were not detected in leaves infected with C. nicotianae. Therefore the two sets of homeologues are regulated in the same way by hormones and respond differently to infection by a pathogen.

Gene structure and expression of a tobacco endochitinase gene in suspension-cultured tobacco cells.

We have isolated and characterized the genomic clone lambda CHN50 corresponding to tobacco basic endochitinase (E.C.3.2.1.14). DNA sequence and blotting analysis reveal that the coding sequence of the gene present on lambda CHN50 is identical to that of the cDNA clone pCHN50 and, moreover, the CHN50 gene has its origin in the progenitor of tobacco, Nicotiana sylvestris. Tobacco basic chitinases are encoded by a small gene family that consists of at least two members, the CHN50 gene and a closely related CHN17 gene which was characterized previously. By northern blot analysis, it is shown that the CHN50 gene is highly expressed in suspension-cultured tobacco cells and the mRNA accumulates at late logarithmic growth phase. To identify cis-DNA elements involved in the expression of the CHN50 gene in suspension-cultured cells, the chimeric gene consisting of 1.1 kb CHN50 5' upstream region fused to the coding sequence of beta-glucuronidase (GUS) was introduced by electroporation into protoplasts isolated from suspension-cultured tobacco cells. Transient GUS activity was found to be dependent on the growth phase of the cultured cells, from which protoplasts had been prepared. Functional analysis of 5' deletions suggests that the distal region between -788 and -345 contains sequences that potentiate the high-level expression in tobacco protoplasts and the region (-68 to -47) proximal to the TATA box functions as a putative silencer.

Structure and expression of a tobacco beta-1,3-glucanase gene.

We determined the primary structure of a tobacco beta-1,3-glucanase gene. The beta-1,3-glucanase gene has a single large intron, and the intron separates coding regions of the signal peptide and the mature enzyme. Analysis of the 5'-flanking region sequence revealed an 11 bp GC-rich element with perfect homology to the putative regulatory sequence of tobacco chitinase genes. RNA blot analysis showed that levels of mRNAs of beta-1,3-glucanase and chitinase are coordinately increased in response to ethylene and salicylic acid. Accumulation of beta-1,3-glucanase mRNA in suspension-cultured cells is rapidly induced at late logarithmic growth phase. Members of the tobacco beta-1,3-glucanase gene families are classified into two subfamilies. One of the subfamilies appeared to be transcriptionally inactive.

Construction of a novel artificial-ribozyme-releasing plasmid.

A novel 'active-ribozyme-releasing system' was constructed, taking advantage of the consensus sequence of a new class of ribozyme. An active ribozyme sequence, targeted for the SFL1 gene (a yeast suppressor gene for flocculation) was fused just downstream of the T7 promoter. The 3' terminus of the first ribozyme was designed to be trimmed by the second ribozyme connected to the downstream of the first active ribozyme. In vitro experiments revealed that the active ribozyme targeted to SFL1 was successfully released by the action of the second ribozyme, subsequently cleaving the SFL1 mRNA at the predetermined site. Since the first active ribozyme with a defined 3'-terminus can be produced even when a circular DNA is used as a template, this kind of construct has a potential to release an 'active ribozyme' tailored to destroy a target gene (RNA) in vivo. Moreover, the second ribozyme in this construct can be utilized as a universal pseudo-terminator for generation of any RNA transcripts inserted in place of the cassette portion of the first ribozyme.

Structure of a tobacco endochitinase gene: evidence that different chitinase genes can arise by transposition of sequences encoding a cysteine-rich domain.

The endochitinases (E.C. 3.2.1.14, chitinase) are a structurally diverse group of enzymes believed to be important in the biochemical defense of plants against potential pathogens. The gene for a chitinase of Nicotiana tabacum L. cv. Havana 425 has been cloned and sequenced. The major transcription start is 11 bp upstream of the ATG codon and 28 bp downstream of the TATA box. The gene contains two introns and encodes a basic chitinase of 329 amino acids with a 23 amino acid N-terminal signal peptide followed by a 43 amino acid, cysteine-rich domain, which is linked by a hinge region to the main structure of the enzyme. This gene appears to be expressed because the exons are identical to the coding sequence of a cDNA which was isolated. Comparison of chitinase amino acid sequences from different plants indicates there are at least three classes of these enzymes: class I, basic chitinases with an N-terminal cysteine-rich domain and a highly conserved main structure; class II, chitinases similar to the main structure of class I chitinases but lacking the cysteine-rich domain; and, class III, chitinases with conserved sequences different from those of the class I and II enzymes. The sequences encoding the cysteine-rich domain in class I chitinases are flanked by 9-10 bp imperfect direct repeats suggesting that these domains arose from a common ancestral gene and were introduced into genes for class I enzymes by transposition events.

In vivo RNA transcript-releasing plasmid possessing a universal pseudo-terminator by means of artificial ribozymes.

RNA transcript-releasing plasmid has been constructed by means of artificial hammerhead ribozymes. In this specific construct of pGENE8459v3 the ribozyme targeted for SFL1 gene (a yeast suppressor gene for flocculation) was fused between two other ribozymes called 5'-processing and 3'-processing ribozymes. Since the "Ribozyme for SFL1" portion (cassette) can be replaced by other RNA sequences, it is now possible to produce any RNAs with defined 5'- and 3'- ends.