Plant Physiol. (1998) 117: 1445-1461

Direct Evidence for Rapid Degradation of Bacillus thuringiensis Toxin mRNA as a Cause of Poor Expression in Plants¹

E. Jay De Rocher, Tracy C. Vargo-Gogola², Scott H. Diehn³, and Pamela J. Green^*

Michigan State University-Department of Energy Plant Research Laboratory (E.J.D.R., S.H.D., P.J.G.), Department of Physiology (T.C.V.-G.), Department of Botany and Plant Pathology (S.H.D.), and Department of Biochemistry (P.J.G.), Michigan State University, East Lansing, Michigan 48824

	ABSTRACT

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It is well established that the expression of Bacillus thuringiensis (B.t.) toxin genes in higher plants is severely limited at the mRNA level, but the cause remains controversial. Elucidating whether mRNA accumulation is limited transcriptionally or posttranscriptionally could contribute to effective gene design as well as provide insights about endogenous plant gene-expression mechanisms. To resolve this controversy, we compared the expression of an A/U-rich wild-type cryIA(c) gene and a G/C-rich synthetic cryIA(c) B.t.-toxin gene under the control of identical 5 and 3 flanking sequences. Transcriptional activities of the genes were equal as determined by nuclear run-on transcription assays. In contrast, mRNA half-life measurements demonstrated directly that the wild-type transcript was markedly less stable than that encoded by the synthetic gene. Sequences that limit mRNA accumulation were located at more than one site within the coding region, and some appeared to be recognized in Arabidopsis but not in tobacco (Nicotiana tabacum). These results support previous observations that some A/U-rich sequences can contribute to mRNA instability in plants. Our studies further indicate that some of these sequences may be differentially recognized in tobacco cells and Arabidopsis.

	INTRODUCTION

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Gene transfer into plants has become a relatively simple and routine process in many species. However, successful integration of a transgene into the plant genome does not automatically result in expression. For example, introduction of an additional copy of an endogenous gene can trigger cosuppression mechanisms, resulting in silencing of both the introduced and the endogenous genes (for review, see Meyer and Saedler, 1996). In the case of foreign genes transferred into plants, it is not uncommon to find that the introduced gene is incompatible with plant gene-expression mechanisms. Bacillus thuringiensis (B.t.)-toxin genes are perhaps the most widely known example of foreign genes for which it has been difficult to obtain useful levels of expression in transgenic plants (Diehn et al., 1996). Although a variety of mechanisms controlling gene expression have been blamed for poor B.t.-toxin expression, the exact cause remains controversial because of the limited amount of data gathered to address the problem. This is in part due to the inherent difficulties associated with studying a gene expressed at very low levels. It is also due to the fact that, for practical applications in agriculture, this problem can be circumvented by the brute-force approach of constructing highly modified synthetic versions of B.t.-toxin genes that yield high levels of expression. Nevertheless, elucidating the mechanisms that limit B.t.-toxin expression could make the design of new genes more efficient and provide insight about steps in gene expression that are relevant to endogenous plant genes.

A widely observed characteristic of B.t.-toxin gene expression in plants has been that little or no mRNA accumulates, even when transcription is under the control of strong promoters. Because mRNA instability provides a simple explanation, it has been widely assumed that B.t.-toxin transcripts are rapidly degraded in plants. However, this has not been clearly demonstrated, and the limited data that are available have led to contradictory conclusions about the potential role of mRNA stability. Instability of B.t.-toxin transcripts in plants was proposed initially to account for the lack of a correlation between promoter activity and mRNA accumulation in early efforts to express B.t.-toxin genes in plants (Fischhoff et al., 1987; Vaeck et al., 1987). In stably transformed tobacco (Nicotiana tabacum) plants, the majority of B.t.-toxin mRNA was present as transcripts that were shorter than full length (Barton et al., 1987). These short transcripts were assumed to be degradation products, and their presence was used to argue that B.t.-toxin mRNA was unstable. However, data were not presented that would distinguish between mRNA degradation and other mechanisms that could produce the short transcripts (such as premature transcription termination, splicing, or polyadenylation). The proposed instability of B.t.-toxin mRNAs was not tested experimentally in these studies.

It has been demonstrated that the construction of synthetic B.t.-toxin genes with highly modified nucleotide sequences can result in high expression in plants (for review, see Diehn et al., 1996). In those cases in which the effect of extensive sequence modifications on mRNA accumulation was examined, it was found that mRNA levels were increased substantially (Perlak et al., 1991; Adang et al., 1993). The design criteria for the synthetic genes has often included sequence changes targeted at potential mRNA instability elements (Perlak et al., 1990, 1991, 1993; Sutton et al., 1992; Adang et al., 1993; van der Salm et al., 1994). Although in all cases the modifications gave higher expression to varying degrees, the effects of the sequence changes on B.t.-toxin mRNA stability were not tested directly by comparing the stability of the transcripts encoded by the synthetic genes and that of corresponding unmodified genes. Because potential mRNA instability elements were only one of several types of targets for sequence modification, it was not possible to attribute the increased expression to a change in B.t.-toxin transcript stability.

The low accumulation of mRNA from wild-type B.t.-toxin genes in plants has presented a technical challenge for determining the role of mRNA stability in poor B.t.-toxin gene expression. Two previous efforts to assess the stability of B.t.-toxin mRNA in plants led to opposite conclusions after the turnover of cryIA(b) B.t.-toxin transcripts in protoplasts was characterized. Murray and coworkers (1991) showed that cryIA(b) transcripts disappeared faster qualitatively than octopine synthase transcripts after electroporation of plasmids carrying the genes into carrot protoplasts. In contrast, van Aarssen and coworkers (1995) concluded that cryIA(b) mRNA was not unstable in plants, based primarily on a comparison of turnover of in vitro-synthesized cryIA(b) and bar transcripts after electroporation into tobacco mesophyll protoplasts. It is unclear why the two groups reached opposite conclusions regarding cryIA(b) mRNA stability, but the differing results may stem from differences between carrot and tobacco and/or the different experimental methods used.

In the present study we used a well-established experimental system to determine the relative half-lives of mRNAs from a wild-type cryIA(c) and a highly modified synthetic cryIA(c) gene. Reliable half-lives are routinely measured for mRNAs transcribed from genes stably integrated into the nuclear genome using tobacco BY-2 cells (Newman et al., 1993; Ohme-Takagi et al., 1993; Taylor and Green, 1995; Sullivan and Green, 1996; van Hoof and Green, 1996). In this report we demonstrate that wild-type cryIA(c) transcripts are degraded more rapidly than synthetic cryIA(c) transcripts in stably transformed BY-2 cells, with a half-life comparable with that of transcripts known to be unstable in plants. Our results provide direct evidence that mRNA stability can be a factor limiting the expression of foreign genes in plants. We also show that the type of modifications typically used to construct highly expressing synthetic B.t.-toxin genes can function at least in part by stabilizing the transcripts in plants. The distribution of the sequence determinants within the wild-type B.t.-toxin transcripts that limit mRNA accumulation in tobacco cells and Arabidopsis plants differs. The differential recognition of these determinants between these two plant systems may have important implications for the engineering of foreign genes for optimal expression in plants and for the understanding of the mechanisms controlling accumulation of mRNA from endogenous genes.

	MATERIALS AND METHODS

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Plant Materials

Arabidopsis ecotype Columbia plants were grown in soil under a 12-h light/12-h dark cycle at 20°C. Transgenic Arabidopsis plants were generated using the vacuum-infiltration method (van Hoof and Green, 1996). Seedlings were grown by germination of seeds on solid Arabidopsis growth medium (Taylor et al., 1993).

Maize (Zea mays cv BMS) cells (a gift from Pioneer Hi-Bred International, Inc., Des Moines, IA) were grown in suspension culture in BMS 237 medium (4.3 g/L Murashige-Skoog salts, 30 g/L Suc, 0.1 g/L myo-inositol, 1.8 mL of 10² M 2,4-D, and 2 mL/L 500× Murashige-Skoog vitamins [Sigma], brought to pH 5.6 with KOH) at 28°C in a rotary shaker at 150 rpm.

Synthetic Bacillus thuringiensis (B.t.)-Toxin Gene Construction

View larger version (24K): [in this window] [in a new window]   Figure 1. Schematic representation of the method used to construct the synthetic B.t.-toxin gene. A two-step PCR approach was used to generate synthetic versions of each of four segments of a cryIA(c) gene using sets of overlapping oligonucleotides that incorporated sequence changes according to the criteria described in the text. As shown for one of the four segments, the alternating sense and antisense polarity of the overlapping modified-sequence oligonucleotides indicated by the directions of the arrows allowed the oligonucleotides to anneal to each other and serve as primers for DNA synthesis in PCR. After the first set of 10 PCR cycles, the addition of 30-mer terminal primers followed by a further 25 PCR cycles preferentially amplified those molecules spanning the full segment. PCR products for each segment were cloned and then assembled by standard cloning methods to generate the complete synthetic B.t.-toxin-coding region.

Thirty-base-pair terminal primers complementary to the ends of PCR products spanning the entire gene segment were used to preferentially amplify the full-length molecules over 25 cycles. PCR products of the correct size were cut out of low-melting-point agarose gels and cloned after removal of terminal adenosines into the EcoRV site of a Bluescript II SK() vector with a modified multiple cloning site (p948) (Diehn et al., 1998). Clones containing inserts were screened by dideoxy sequencing (Sanger et al., 1977) for PCR products with no or only one to two sequence errors. Sequencing was done with a 7-deaza-dGTP Sequenase sequencing kit (United States Biochemical) to overcome compression artifacts caused by the high G/C content of the modified sequence.

For those synthetic segments for which no clone was found to be entirely free of sequence errors, in vitro mutagenesis (Kunkel et al., 1987) was used to introduce the correct sequence. All segments were then assembled in pUC118 by standard cloning methods using the restriction sites shown in Figure 1 to generate the complete synthetic cryIA(c)-coding region (p1334). The synthetic coding region was sequenced on both strands to verify that the sequence was correct. The nucleotide sequence of the synthetic gene has been deposited in the GenBank Sequence Database. The modified sequence oligonucleotides are described in the annotation section of the GenBank entry. The frame-shift synthetic B.t.-toxin gene described in the text resulted from insertion of a single cytosine 498 bp downstream of the adenosine of the translational start ATG codon (p1329).

Gene Constructions

View larger version (24K): [in this window] [in a new window]   Figure 2. Schematic diagram of B.t.-toxin and control gene constructions. Expression of wild-type B.t.-toxin, synthetic B.t.-toxin, and globin genes in transient expression assays and stably transformed cell lines and plants was under the control of 2X35S. For transient expression in maize, constructs included an ADH1 intron. Polyadenylation signals were provided by the 3 UTR from the pea Rubisco small subunit rbcS-E9 gene in all constructs. Plasmid numbers for each construct are indicated at the left.

Similar plasmid constructions were used for transient expression in maize BMS cells but included an ADH1 intron inserted between the promoter and the coding region. For transient expression of the synthetic cryIA(c) gene in BMS cells, the cryIA(c)-coding region and rbcS-E9 3 UTR contained in a fragment excised from p1335 with BglII and ClaI were inserted into p1138 (Diehn et al., 1998) containing the 2X35S and ADH1 intron cut with BamHI and ClaI to yield p1345 (Fig. 2). The wild-type cryIA(c)-coding region and rbcS-E9 3 UTR were excised from p995 (Diehn et al., 1998) with BbuI and BamHI and substituted for the synthetic cryIA(c)-coding region in p1345 cut with BbuI and BamHI to yield p1346 (Fig. 2). To generate the globin gene construction for use in BMS cells (p1160), the human -globin-coding region was excised with NcoI and XbaI from the previously described plasmid p962 (Newman et al., 1993) and inserted into the EcoRV site of a Bluescript II SK() vector with a modified multiple cloning site (p948) to yield p963. The -globin-coding region was then excised from p963 with XbaI and BamHI and substituted for the GUS-coding region in p840, a pUC11 derivative containing a CaMV 35S-GUS-E9 expression construct (details on request) cut with XbaI and BamHI to yield p977 containing the expression cassette CaMV 35S-globin-E9. The -globin-coding region and rbcS-E9 3 UTR were excised with BglII and ClaI from p977 and inserted into p1138 cut with BamHI and ClaI to yield p1160 (Fig. 2) containing the expression cassette 2X35S-ADH1 intron-globin-E9.

For transformation into tobacco and Arabidopsis, the wild-type and synthetic 2X35S-cryIA(c)-E9 expression cassettes were transferred to the unique HindIII site in pBI121 as HindIII fragments excised from p1204 and p1335 to yield p1205, containing the wild-type cryIA(c), and p1410, containing the synthetic cryIA(c). The direction of transcription of the cryIA(c) genes was the same as that of the GUS gene in the pBI121 plasmid.

Chimeric wild-type/synthetic cryIA(c) genes were constructed by substituting wild-type segments for the corresponding segments in the synthetic gene. Wild-type segment 1 was excised from p1310, a pUC118 plasmid containing only the wild-type cryIA(c)-coding region, with BbuI and XbaI and inserted into p1334 cut with BbuI and XbaI, yielding p1405. Synthetic segment 2 was added back as an XbaI fragment from p1334 by insertion into p1405 cut with XbaI to give p1440 (wt1-syn2-syn3-syn4). Wild-type segment 2 was excised from p1310 as an XbaI fragment and inserted into p1334 cut with XbaI to yield p1406 (syn1-wt2-syn3-syn4). Wild-type segment 3 was excised with XbaI and AccI from p1310 and inserted into p1334 cut with XbaI and AccI to yield p1407. Synthetic segment 2 was added back as an XbaI fragment from p1334 by insertion into p1407 cut with XbaI to yield p1442 (syn1-syn2-wt3-syn4). Wild-type segment 4 was excised from p1310 AccI and BamHI and inserted into p1334 cut with AccI and BamHI to yield p1411 (syn1-syn2-syn3-wt4). To assemble expression cassettes, all four chimeric coding regions were excised with BbuI and BamHI and substituted for the synthetic cryIA(c)-coding region in p1335 cut with BbuI and BamHI to yield p1450, p1451, p1452, and p1454 containing wild-type segments 2, 4, 1, and 3, respectively, in the chimeric coding regions. For transformation into BY-2 cells and Arabidopsis plants, transformation constructions were made by excision of the four expression cassettes as HindIII fragments and insertion into the HindIII site in pBI121 in the same transcriptional orientation as the GUS gene in pBI121 to give p1505, p1506, p1507, and p1508 containing wild-type segments 1, 2, 3, and 4, respectively, in the chimeric cryIA(c)-coding regions.

Electroporation of Tobacco and Maize Protoplasts

Maize BMS protoplasts were prepared and electroporated by the same method but with the following substitutions. NT wash solution was replaced with BMS wash solution (250 mM mannitol, 50 mM CaCl₂, and 5 mM Mes, brought to pH 5.5 with KOH). NT electroporation buffer was replaced with BMS electroporation buffer (200 mM mannitol, 120 mM KCl, 10 mM NaCl, 4 mM CaCl₂, and 10 mM Hepes, brought to pH 7.2 with NaOH). NT plating medium was replaced with BMS plating medium (80 mL of BMS 237 medium, 20 mL of BMS culture supernatant, and 300 mM mannitol, brought to pH 5.6 with KOH). Electroporator settings were 250 V and 960 µFD.

RNA Isolation

RNA Gel Blots

DNA Probes

Half-Life Determination

Protein Analysis

Insect Bioassays

For bioassays of insecticidal activity in transgenic Arabidopsis, T₂ seeds were pooled from 16 lines expressing the wild-type B.t.-toxin gene and 16 lines expressing the synthetic B.t.-toxin gene. Pooled transgenic seeds and untransformed seeds were sterilized in 50% bleach solution for 7 min, washed in sterile water, and then plated on primary-selection Arabidopsis growth medium plates (4.3 g/L Murashige-Skoog salts, 1× B5 vitamins, 1% [w/v] Suc, 0.5 g/L Mes, and 0.8% Phytagar, brought to pH 5.7 with KOH) without drugs for germination. After 2 weeks, approximately 100 seedlings were transferred to 150- × 15-mm plates containing two layers of Whatman paper (Fisher Scientific) dampened with sterile water. Within 24 h after hatching, 15 tobacco hornworms were transferred to each of the plates and kept under continuous light at 27°C for the course of the assay.

Isolation of Nuclei from BY-2 Cells

Nuclear Run-On Assays

	RESULTS

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Design and Construction of a Synthetic B.t.-Toxin Gene Incorporating Modifications That Enhance Expression in Plants

It has been demonstrated for several B.t.-toxin genes that extensive modification of the coding-region nucleotide sequence can result in increased expression (for review, see Diehn et al., 1996). The A/T richness typical of B.t.-toxin genes tends to occur at the third position of codons, and codons with A or T in the third position tend to be rarely used in plants. Therefore, we chose a strategy that improved the codon usage and simultaneously eliminated the A/T richness. Specifically, we introduced a codon bias typical of maize genes (Wada et al., 1992) without altering the original amino acid sequence. This codon bias was chosen in part because of the extensive data that were available on codon usage in maize genes and the importance of maize and other cereals for agriculture. Moreover, because the codon bias in monocot genes tends to be more stringent than in dicot genes and the set of preferred codons in maize is a subset of those typically used in dicots (Campbell and Gowri, 1990), the codon bias introduced into the B.t.-toxin gene would be compatible with expression in dicot as well as in monocot species.

The strong preference for G or C in the third position in maize genes had the benefit of eliminating by default A/T-rich sequences that might be recognized in plants as unwanted signals for RNA processing and turnover. Other design criteria were to preserve several restriction sites that would be useful in the construction of the synthetic gene, to avoid introducing restriction sites that would interfere with construction or cloning steps, and to avoid introducing A/T-rich sequences and theoretically stable secondary structures. Table I summarizes the results of the sequence modifications and shows that sequence changes were distributed uniformly throughout the coding region. Of 610 codons, 496 were altered, resulting in an increase in G/C content from 38% to 64%.

After a modified sequence meeting all of the above criteria was designed, the synthetic B.t.-toxin gene was constructed using the PCR-based approach shown in Figure 1. The modified-sequence oligonucleotides ranged in size from 81 to 132 bases in length. Synthesis of segments 1 and 2 was done using four overlapping oligonucleotides (Fig. 1). Segments 3 and 4 were generated using sets of six and eight overlapping oligonucleotides, respectively, because of the greater lengths of these segments. PCR products were cloned and sequenced to identify synthetic B.t.-toxin gene segments with the fewest or no sequence errors. Where necessary, sequence errors were corrected by in vitro mutagenesis. After assembly of all segments, the complete synthetic coding region was resequenced on both strands to verify that the sequence was correct.

Differential mRNA Accumulation Resulting from Sequence Modifications Incorporated into the Synthetic B.t.-Toxin Gene

View larger version (49K): [in this window] [in a new window]   Figure 3. RNA gel-blot analysis of relative expression levels of the synthetic and wild-type B.t.-toxin genes in plant cells. Accumulation of synthetic (SYN) and wild-type (WT) B.t.-toxin mRNAs was compared in transiently transformed maize and tobacco cells and in stably transformed tobacco cells and Arabidopsis plants. A, Plasmids containing wild-type or synthetic B.t.-toxin genes were electroporated into maize BMS and tobacco BY-2 protoplasts. Plasmids containing a human -globin gene were coelectroporated in both types of experiments to serve as an internal standard. The positions of the B.t.-toxin and globin transcripts are indicated. B, Tobacco BY-2 cells and Arabidopsis plants were stably transformed with constructs containing wild-type or synthetic B.t.-toxin genes and a GUS gene serving as an internal standard. The positions of the B.t.-toxin and GUS transcripts are indicated.

Wild-type and synthetic B.t.-toxin gene expression was also assessed in stably transformed BY-2 cells because this cell line is well established as a system for the measurement of mRNA half-lives (Newman et al., 1993) and could also be used for measuring relative transcription rates of the two genes. Wild-type and synthetic B.t.-toxin genes driven by 2X35S were inserted into pBI121 vector and introduced into BY-2 cells by A. tumefaciens-mediated transformation. The CaMV 35S-driven GUS gene present in the pBI121 plasmid produces a relatively stable transcript in plants (Newman et al., 1993) and served as a co-transformed internal standard for comparison of the expression of the two B.t.-toxin genes in independently transformed cell lines. Kanamycin-resistant calli were screened histochemically for GUS expression, and the calli from more than 90 independent GUS-positive lines were pooled for RNA extraction. Independent lines were pooled to average out possible variations in expression levels among individual lines arising from differences in transgene copy number and from position effects on transgene transcriptional activity as a result of different sites of genomic integration. The left panel in Figure 3B shows a representative RNA gel blot of pools of BY-2 cell lines stably transformed with the wild-type or synthetic B.t.-toxin genes. As in electroporated protoplasts, wild-type B.t.-toxin transcripts were undetectable, whereas accumulation of the transcripts encoded by the synthetic B.t.-toxin gene was comparable with that of transcripts from the internal reference gene.

To verify that the expression pattern seen in transiently transformed protoplasts and stably transformed cell lines was representative of intact plants, we attempted to introduce both B.t.-toxin genes into tobacco plants by A. tumefaciens-mediated transformation (Newman et al., 1993). Shoots regenerated on kanamycin-containing medium were screened histochemically for GUS activity. Although nearly all plants regenerated from the transformation with the wild-type B.t.-toxin gene were GUS positive, almost no plants regenerated from the synthetic B.t.-toxin transformation had detectable GUS activity. The few plants that were GUS positive exhibited no detectable B.t.-toxin transcript accumulation in RNA gel-blot analysis (data not shown). Because initial shoot formation was noticeably delayed for plants regenerated from the synthetic B.t.-toxin transformation, most of these plants were likely to be escapes or to have low transgene expression due to position effects. Repeated transformation of both constructs yielded similar results.

To rule out the possibility of a defect in the synthetic B.t.-toxin transformation construct, the plasmid was rebuilt and the transformation procedure repeated for the wild-type and synthetic genes, with similar results. Parallel transformations with a third construct containing a synthetic B.t.-toxin gene with a frame-shift mutation yielded GUS-positive plants as readily as transformations done with the wild-type B.t.-toxin gene. The frame-shifted synthetic B.t.-toxin gene was recovered during the construction of the synthetic gene, and was the result of a single C insertion, resulting in a premature stop codon about one-third of the way into the coding region. This premature stop codon would prevent expression of an insecticidally active B.t.-toxin protein, since the portion of the protein that would remain untranslated is known to be necessary for activity (Fischhoff et al., 1987).

Because the frame-shifted and synthetic B.t.-toxin transformation constructs were identical except for a single C insertion and the frame-shifted construct could not produce active B.t. toxin, the difference in transformation results may be indicative of a toxic effect of highly expressed active B.t.-toxin protein in regenerating tobacco cells. In contrast, expression of the synthetic B.t.-toxin gene in tobacco calli and suspension cultures had no discernible deleterious effect.

To determine whether this problem was specific for tobacco plants, the same wild-type and synthetic B.t.-toxin transformation constructs were introduced into Arabidopsis by A. tumefaciens-mediated transformation using vacuum infiltration (van Hoof and Green, 1996). The right panel of Figure 3B shows a gel blot of RNA extracted from leaves pooled from 15 independent kanamycin-resistant lines for each construct. As seen in protoplasts and stably transformed tobacco cells, wild-type B.t.-toxin mRNA was undetectable, but synthetic B.t.-toxin mRNA accumulated to an easily detected level. The recovery of Arabidopsis plants expressing the synthetic B.t.-toxin gene indicates that the problem encountered with generation of transgenic tobacco plants is not a general one.

The sequence changes incorporated into the synthetic B.t.-toxin gene substantially increased mRNA accumulation over the wild-type B.t.-toxin gene in protoplasts and in stably transformed tobacco cell lines and Arabidopsis plants. It was not possible to quantitate the magnitude of the increase, because wild-type B.t.-toxin transcripts did not accumulate to detectable levels. However, RNA gel-blot analysis performed on a dilution series of RNA from cells expressing the synthetic B.t.-toxin gene was able to give a minimum estimate of the magnitude of increase by determining how many times the hybridization signal could be diluted before decreasing to background levels. Figure 4 shows the results of such an experiment using total RNA from a transient-expression experiment in tobacco protoplasts that produced the highest hybridization signals above background. The synthetic B.t.-toxin hybridization signal was still detectable above background at a 16-fold dilution, indicating that B.t.-toxin mRNA accumulation was improved at least 16-fold by the sequence modifications. Other experiments with overloaded gels indicated that the difference was at least 64-fold (data not shown).

View larger version (48K): [in this window] [in a new window]   Figure 4. Estimation of the minimum difference in mRNA accumulation between wild-type (WT) and synthetic (SYN) B.t.-toxin genes. Tobacco BY-2 protoplasts were electroporated with plasmids containing either wild-type or synthetic B.t.-toxin genes and plasmids containing a human -globin gene as an internal standard. RNA from protoplasts expressing the synthetic B.t.-toxin gene was diluted in 2-fold steps up to 64-fold with total RNA from untransformed tobacco cells. Each lane was loaded with 20 µg of RNA. The positions of the B.t.-toxin and globin transcripts are indicated by arrowheads. The blot was hybridized with a probe specific for the rbcS-E9 3 UTR common to all three transcripts. Dilution factors are indicated by numbers above the lanes.

High-Level Accumulation of Functional Protein Produced by the Synthetic B.t.-Toxin Gene

View larger version (60K): [in this window] [in a new window]   Figure 5. Accumulation of B.t.-toxin protein in plants expressing the wild-type and synthetic B.t.-toxin genes as determined by SDS-PAGE analysis of protein extracts from pools of independent Arabidopsis lines stably transformed with the wild-type (WT) or synthetic (SYN) B.t.-toxin genes. Purified cryIA(c) protein (predicted molecular mass approximately 133 kD) expressed in E. coli was included as a positive control (C) for anti-cryIA(c) antibody binding. Positions of molecular mass standards (in kD) are indicated between the panels. A, Immunoblot incubated with anti-cryIA(c) polyclonal antibodies. B, Immunoblot of duplicate lanes incubated with anti-cryIA(c) polyclonal antibodies and overdeveloped to demonstrate the lack of detectable B.t. toxin in plants transgenic for the wild-type B.t.-toxin gene.

The functionality of the protein expressed in stably transformed Arabidopsis seedlings and BY-2 cells was assessed in bioassays of insecticidal activity against tobacco hornworm. For bioassays of Arabidopsis, hornworm larvae were given as a sole food source seedlings pooled from 16 independent lines transgenic for either the wild-type or synthetic B.t.-toxin genes. Untransformed wild-type seedlings were used as a control for the toxic effects of Arabidopsis seedlings. No hornworms survived on Arabidopsis seedlings expressing the synthetic B.t.-toxin gene 3 d after initiation of feeding, as shown in Table II. Survival was 100% for hornworms feeding on Arabidopsis seedlings expressing the wild-type B.t.-toxin gene or on untransformed seedlings.

To examine B.t.-toxin insecticidal activity in BY-2 cells, crude extracts of stably transformed cell lines expressing either the wild-type or the synthetic B.t.-toxin genes were applied to the surfaces of plates containing solid tobacco hornworm medium. Crude extracts of untransformed BY-2 cells were used as a control for any toxicity of the BY-2 cells. Extracts from all cell lines expressing the synthetic B.t.-toxin gene resulted in almost no surviving hornworms. The effect of extract from the wild-type gene WT1 BY-2 line was indistinguishable from that of extract from an untransformed BY-2 cell line, with 100% survival observed. A second BY-2 line expressing the wild-type gene WT2 exhibited a higher but still very low level of B.t.-toxin mRNA accumulation than the WT1 cell line in RNA gel-blot analysis (data not shown), which was likely the result of positional effects. Extract from the WT2 line resulted in almost no hornworm survival, demonstrating that even low-level expression of a wild-type B.t.-toxin gene can give insecticidal activity against a sensitive species such as tobacco hornworm, as observed previously (Fischhoff et al., 1987; Delanney et al., 1989). The results of the bioassays demonstrated, as expected, that the product of the synthetic B.t.-toxin gene has marked insecticidal activity.

Relative Transcriptional Activities of Wild-Type and Synthetic B.t.-Toxin Genes

As shown in Table III, the average ratios of wild-type and synthetic B.t.-toxin transcriptional activities to GUS-transcriptional activity were nearly identical (0.56 ± 0.16 and 0.59 ± 0.13, respectively). The use of the rbcS-E9 3 UTR ensured that only transcription products extending through the full coding region and into the 3 UTR were detected, and that products of premature termination would not contribute to the B.t.-toxin hybridization signals. The similarity in the transcriptional activities of the wild-type and synthetic genes indicates that the wild-type B.t.-toxin-coding region is not likely to contain sequences that block transcription initiation, stall transcription, or cause premature termination. Although we cannot formally exclude the possibility that these processes occur but the effects are somehow canceled out, it is clear that the net transcriptional activities of the wild-type and synthetic B.t.-toxin genes are the same. These data indicate that the large difference in mRNA accumulation between the two genes is attributable to a posttranscriptional step in the expression of the wild-type B.t.-toxin gene in plants.

Half-Lives of Wild-Type and Synthetic B.t.-Toxin mRNAs in Plants

BY-2 cells expressing the wild-type B.t.-toxin gene were treated transiently with CHX to generate levels of wild-type B.t.-toxin mRNA accumulation sufficient for detection over a standard half-life time-course experiment after removal of the CHX. Successful application of this approach was recently reported for mRNA half-life measurement in yeast (Zhang et al., 1997). Inhibition of translation with CHX could stabilize transcripts by causing the rapid loss of a labile protein factor that normally destabilizes the mRNA in trans. Alternatively, stabilization could result from blocking a cis-acting mRNA turnover mechanism that requires ongoing translation of the transcript. We determined an optimal minimum concentration of CHX for which the effect on mRNA accumulation was rapidly reversible but still sufficient to make measurement of mRNA half-lives practical (data not shown). CHX had no effect on the accumulation of synthetic B.t.-toxin mRNA, consistent with the possibility that the sequence modifications incorporated in the synthetic gene eliminated instability determinants.

Independent BY-2 cell lines transformed with the wild-type B.t.-toxin gene were screened by RNA gel-blot analysis to identify individual lines with the highest wild-type B.t.-toxin mRNA accumulation in response to CHX treatment (data not shown). Most cell lines accumulated wild-type B.t.-toxin mRNA to barely detectable levels after CHX treatment. Differences in mRNA accumulation between lines were probably caused by position effects. The three lines with the greatest accumulation of wild-type B.t.-toxin mRNA after CHX treatment and three independent cell lines expressing the synthetic B.t.-toxin gene were chosen for time-course experiments. Suspension cultures of these cell lines were treated for 2 h with CHX followed by removal of CHX. Transcription was blocked with ActD (Newman et al., 1993), and cells were harvested at intervals over a 2-h time course. For the sake of maintaining consistent experimental conditions, half-life measurements for synthetic B.t.-toxin mRNA were made with the same transient CHX treatment used for measurement of wild-type mRNA half-life. It should be noted, however, that synthetic B.t.-toxin mRNA half-life was unaffected by CHX, as determined by half-life measurements made with and without CHX treatment (data not shown). The accumulation and half-life of GUS mRNA, which served as the internal reference, was similarly unaffected by CHX.

The results of RNA gel-blot analysis of time-course experiments are shown in Figure 6. The effect of the transient CHX treatment on wild-type B.t.-toxin mRNA accumulation can be seen by comparing the C and 0 lanes in Figure 6A. In contrast, CHX treatment had no effect on mRNA accumulation of either the synthetic B.t.-toxin or GUS mRNAs (C and 0 lanes in Fig. 6B and lower part of Fig. 6A). The disappearance of wild-type B.t.-toxin mRNA during the time course was visibly faster than that of the GUS mRNA (Fig. 6A). Short transcripts (Fig. 6A) resulting from polyadenylation within the coding region (Diehn et al., 1998) also diminished in amount faster than the GUS transcripts. In contrast, synthetic B.t.-toxin mRNA (Fig. 6B) disappeared at a rate similar to that of the GUS mRNA. Hybridization signals were quantitated and subjected to linear-regression analysis to calculate mRNA half-lives. Of the three lines expressing the wild-type B.t.-toxin gene initially selected for half-life analysis, the hybridization signals obtained in time-course experiments for one of these lines proved to be too close to background for acceptable quantitation. Two independent half-life determinations were made for each of the remaining two cell lines expressing the wild-type B.t.-toxin gene. Synthetic B.t.-toxin mRNA half-life determinations were made for three independent cell lines. All time-course RNA gel blots were reprobed to determine the GUS mRNA half-life in each cell line.

View larger version (44K): [in this window] [in a new window]   Figure 6. Measurement of relative half-lives of wild-type and synthetic B.t.-toxin transcripts (BT). Wild-type and synthetic B.t.-toxin mRNA decay was measured over a time course in stably transformed tobacco BY-2 suspension cell cultures. Cells were harvested before transient CHX treatment (C), after the 2-h treatment followed by removal of CHX (0), at intervals after the addition of ActD (15, 30, 45, 60, 90, and 120 min), and after continuous treatment with CHX and no ActD (+). A, RNA gel-blot analysis of a time-course experiment using a cell line expressing the wild-type B.t.-toxin gene. Full-length wild-type B.t.-toxin transcripts are indicated by the arrowhead, and short polyadenylated B.t.-toxin transcripts are indicated by dark circles. The blot was stripped and rehybridized with a probe specific for GUS mRNA. B, RNA gel-blot analysis of a time-course experiment using a cell line expressing the synthetic B.t.-toxin gene. Full-length synthetic B.t.-toxin transcripts are indicated by the arrowhead. The blot was stripped and rehybridized with a probe specific for GUS mRNA.

The absolute half-lives of wild-type and synthetic B.t.-toxin mRNAs were distinctly different, as can be seen in Table IV. As described above, the genes encoding these transcripts have identical flanking sequences and differ only in the sequences of their coding regions. Wild-type B.t.-toxin transcripts were turned over more rapidly than synthetic B.t.-toxin transcripts, with an average absolute half-life of 19.5 min compared with 66 min. We have found in previous studies that half-lives of different mRNAs can be compared most reliably when expressed as relative half-lives using the half-lives of a reference mRNA common to all cell lines as an internal standard (Newman et al., 1993; Ohme-Takagi et al., 1993). The relative half-life of 0.33 min for the wild-type B.t.-toxin transcripts using GUS as the reference transcript places them among the most rapidly degraded mRNAs for which measurements have been made in BY-2 cells (Newman et al., 1993; Taylor and Green, 1995; Gil and Green, 1996; Sullivan and Green, 1996). After normalization to GUS mRNA half-life, the relative turnover rate of wild-type B.t.-toxin transcripts was approximately 3.6 times faster than that of synthetic B.t.-toxin transcripts, demonstrating that the sequence modifications included in the synthetic gene resulted in transcript stabilization.

Distribution of Sequences in the Wild-Type B.t.-Toxin Gene That Limit mRNA Accumulation in Plants

View larger version (25K): [in this window] [in a new window]   Figure 7. Effects of wild-type B.t.-toxin sequences on synthetic B.t.-toxin mRNA accumulation. A, Schematic diagram of chimeric gene constructs used to test the effects of wild-type B.t.-toxin sequences on mRNA accumulation. The synthetic B.t.-toxin-coding region was divided into four segments using restriction sites conserved between the wild-type and synthetic coding regions. Each segment of the wild-type gene was substituted individually for the corresponding segment of the synthetic gene. B, Histogram summarizing the results of RNA gel-blot analysis of chimeric B.t.-toxin gene expression relative to the synthetic B.t.-toxin gene in pools of stably transformed tobacco BY-2 cell lines (white bars) and Arabidopsis plants (black bars). Hybridization signals for full-length B.t.-toxin mRNAs were quantitated and normalized using GUS-hybridization signals as an internal standard. Hybridization signals of the synthetic B.t.-toxin transcripts in tobacco and Arabidopsis were set equal to one to allow comparison of relative expression levels between tobacco and Arabidopsis. Data are presented as means and SEs for three sets of tobacco and four sets of Arabidopsis pools.

The effect of wild-type B.t.-toxin sequences on synthetic B.t.-toxin mRNA accumulation was used as an indicator of the presence of detrimental sequence elements in the wild-type segments. For tobacco, three independent pools containing at least 50 independent transformed BY-2 cell lines were made for each of the chimeric constructs. For Arabidopsis, four independent pools containing seedlings from 10 to 15 independent lines were made for each construct. Relative mRNA accumulation levels for each of the chimeric B.t.-toxin genes were determined by gel-blot analysis of RNA isolated from the pools of BY-2 and Arabidopsis lines. Hybridization signals for full-length chimeric B.t.-toxin transcripts were quantitated and normalized to synthetic B.t.-toxin mRNA accumulation using GUS-hybridization signals as an internal standard.

To allow comparison of relative expression levels of the chimeric genes between tobacco and Arabidopsis, hybridization signals of the synthetic B.t.-toxin transcripts in tobacco and Arabidopsis were each set equal to one. As shown in Figure 7, when substituted for the corresponding segment of the synthetic B.t.-toxin gene, each of the four segments of the wild-type B.t.-toxin gene caused lowered mRNA accumulation relative to the full synthetic gene in Arabidopsis. In contrast, only the 3rd and 4th wild-type gene segments caused reduced chimeric mRNA accumulation in tobacco cells. Segment 4 had the greatest effect, decreasing mRNA accumulation approximately 10-fold relative to the synthetic gene in both Arabidopsis and tobacco. None of the segments reduced the mRNA levels to below the limit of detection, indicating that the very low mRNA levels from the full wild-type B.t.-toxin gene resulted from combined effects of sequence elements present in different segments of the gene. Sequences in wild-type B.t.-toxin gene segments 1 and 2 that reduced chimeric gene mRNA accumulation by 70% to 80% in Arabidopsis were relatively ineffective in tobacco cells, indicating possible species-specific differences in recognition of sequence elements affecting mRNA accumulation. Alternatively, the observed differences may reflect differences in sequence-element recognition in cultured cells and whole plants.

Based on the identification of three polyadenylation sites within the wild-type B.t.-toxin-coding region described in the accompanying report (Diehn et al., 1998), it could be expected that products of aberrant polyadenylation might also be observed in the chimeric genes if segment junctions do not interrupt the regulatory sequences. Short transcripts consistent in size with polyadenylation at the three identified sites were observed for the chimeric genes containing wild-type segments 2, 3, and 4 in both tobacco and Arabidopsis (data not shown). The chimeric gene containing wild-type segment 3 produced small amounts of additional short transcripts that were the same in both tobacco and Arabidopsis and were not analyzed further. Although the accumulation of the chimeric transcript containing segment 4 was quite low, as mentioned above, most of the mRNA produced in both tobacco and Arabidopsis was consistent with polyadenylation at the segment 4 poly(A⁺) site mapped in the accompanying paper (Diehn et al., 1998). The similarity in transcript processing in both species suggests that the difference in the effects of segments 1 and 2 in tobacco and Arabidopsis may be primarily attributable to differential recognition of instability determinants. In contrast to polyadenylation signals, the differences in the effect of some wild-type B.t.-toxin sequences on mRNA accumulation in tobacco and Arabidopsis indicate that at least some sequence elements detrimental to mRNA accumulation may be recognized differently among dicots. This differential recognition, together with the inability to express the synthetic B.t.-toxin gene in transgenic tobacco plants in contrast to transgenic Arabidopsis, argues that there may be inherent differences between species that are relevant to optimizing the expression of B.t.-toxin genes.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Even though the incompatibility of wild-type B.t.-toxin genes and plant gene-expression mechanisms has been known for more than 10 years, and speculation on the potential causes has been extensive, the mechanistic basis for the incompatibility has remained unknown. Extensive sequence modifications have been used in the construction of synthetic B.t.-toxin genes to alleviate the incompatibility. These changes have resulted in high protein and mRNA accumulation without providing any direct information about why the wild-type genes were so poorly expressed to begin with. In this study we show that wild-type B.t.-toxin sequences do not inhibit transcriptional activity. Moreover, we provide the first direct evidence to our knowledge for rapid degradation of wild-type B.t.-toxin mRNA in stably transformed plant cells and show that the sequence modifications typically used to obtain high expression with synthetic B.t.-toxin genes increase transcript stability. In the accompanying paper (Diehn et al., 1998), we also demonstrate for the first time to our knowledge that a wild-type B.t.-toxin-coding region contains sequences that are recognized as polyadenylation signals in plants, thereby interfering with expression of full-length B.t.-toxin mRNAs.

A Synthetic B.t.-Toxin Gene Designed for Use as a Comparative Control and as a Reporter Gene

One approach used in the design of synthetic B.t.-toxin genes has been to modify A/T-rich sequences resembling known sequences involved in R