Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site.

The active site of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) has been probed using site-directed mutagenesis and inhibitor binding techniques. Replacement of a specific glycyl with an alanyl or a prolyl with a seryl residue in a highly conserved region confers glyphosate tolerance to several bacterial and plant EPSPS enzymes, suggesting a high degree of structural conservation between these enzymes. The glycine to alanine substitution corresponding to Escherichia coli EPSPS G96A increases the Ki(app) (glyphosate) of petunia EPSPS 5000-fold while increasing the Km(app)(phosphoenolpyruvate) about 40-fold. Substitution of this glycine with serine, however, abolishes EPSPS activity but results in the elicitation of a novel EPSP hydrolase activity whereby EPSP is converted to shikimate 3-phosphate and pyruvate. This highly conserved region is critical for the interaction of the phosphate moiety of phosphoenolpyruvate with EPSPS.

Agrobacterium-mediated transformation of Solanum tuberosum L. cv. 'Russet Burbank'.

Stem sections from shoot cultures maintained in vitro were used to produce transgenic plants of the potato, Solanum tuberosum L. cv. 'Russet Burbank'. Stem internode pieces inoculated with Agrobacterium tumefaciens containing coat protein genes from potato virus X and potato virus Y, produced shoots with a frequency of 60% in the absence of selection and 10% on medium containing 100 mg/l kanamycin monosulfate. Regenerated shoots were assayed for kanamycin resistance by placing stem segments on callus induction medium containing an increased level of kanamycin. Of a total 255 regenerated shoots, 47 (18%) were kanamycin resistant. Of the kanamycin resistant shoots, 25 (53%) expressed the PVX or PVY coat protein genes as assayed by enzyme-linked immunosorbent assay or Western immunoblot analysis.

Tissue-specific expression of the TMV coat protein in transgenic tobacco plants affects the level of coat protein-mediated virus protection.

Transgenic tobacco plants were produced that express a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) under the control of the promoter from a ribulose bisphosphate carboxylase small subunit (rbcS) gene. Plant lines expressing comparable levels of CP from the rbcS and cauliflower mosaic virus 35S promoters were compared for resistance to TMV. In whole plant assays the 35S:CP constructs gave higher resistance than the rbcS:CP constructs. On the other hand, leaf mesophyll protoplasts isolated from both plant lines were equally resistant to infection by TMV. This indicated that the difference in resistance between the lines in the whole plant assay reflects differences at the level of short- and/or long-distance spread of TMV. Therefore, we propose that the difference in tissue-specific expression between the 35S and rbcS promoters accounts for greater resistance in the plant lines that express the 35S:CP chimeric genes.

Identification and characterization of stamen- and tapetum-specific genes from tomato.

Differential screening of a tomato cDNA library produced from pre-anthesis stamens resulted in the isolation of 25 cDNA clones that hybridized to probes made from stamen RNA and showed no hybridization to probes made from RNA of vegetative organs. The 25 clones were found to represent 11 noncross-hybridizing classes. The majority of these clones were derived from genes that were single or low copy in the tomato genome. Northern RNA blotting experiments of vegetative and floral organs at several stages of development demonstrated that expression in all 11 classes was confined to floral organs. Of the 11 classes 9 were found to be expressed exclusively in stamens prior to anthesis. Two classes showed expression in immature stamens and in petals, with one of these two additionally being expressed in mature stamens at anthesis. Clones from three of the classes that were expressed exclusively in stamens were used as probes for in situ localization of RNA in floral organs. These experiments demonstrated that expression of the genes corresponding to these clones was confined to the tapetal cells of the anthers. Expression of one of the three genes was found to be limited to a single cell type during the 5-6 day period from late meiosis to immature pollen formation.

Protection against tobacco mosaic virus infection in transgenic plants requires accumulation of coat protein rather than coat protein RNA sequences.

Transgenic tobacco plants which express a chimeric gene encoding the tobacco mosaic virus (TMV) coat protein (CP) and the TMV 3' untranslated region are protected against infection by TMV. In this study chimeric genes that encode the sequences representing the TMV CP subgenomic RNA, but do not produce protein (because of removal of the initiation codon), and RNA that lacks the tRNA-like sequence of the TMV 3' end were expressed in transgenic plants. Only plants that accumulated CP, regardless of the presence of absence of the 3' end of TMV-RNA, were protected against infection by TMV. The results indicate that the CP per se, rather than TMV RNA, is responsible for the resistance to infection by TMV. Furthermore, the degree of protection is dependent upon the level of accumulated CP.

Genetically engineering plants for crop improvement.

Dramatic progress has been made in the development of gene transfer systems for higher plants. The ability to introduce foreign genes into plant cells and tissues and to regenerate viable, fertile plants has allowed for explosive expansion of our understanding of plant biology and has provided an unparalleled opportunity to modify and improve crop plants. Genetic engineering of plants offers significant potential for seed, agrichemical, food processing, specialty chemical, and pharmaceutical industries to develop new products and manufacturing processes. The extent to which genetically engineered plants will have an impact on key industries will be determined both by continued technical progress and by issues such as regulatory approval, proprietary protection, and public perception.

Isolation of Tissue-Specific cDNAs from Tomato Pistils.

We have used a differential plaque hybridization screening procedure to isolate cDNA clones for genes that show elevated or exclusive expression in tomato pistils. Clones that showed maximal expression in immature pistils (premeiotic to early meiosis) and mature pistils (at anthesis) were isolated. Of nine clones that were characterized, four were found also to express at some stage of anther development. In situ hybridization experiments showed that expression of the genes we have identified is very tightly regulated both spatially and temporally within the pistil. One gene was identified that is expressed in the pistil only in the transmitting tissue of the style. A second gene was found to express exclusively in two to three cell layers of the ovules for a period of less than eight days.

In vitro transcription and translational efficiency of chimeric SP6 messenger RNAs devoid of 5' vector nucleotides.

A plasmid containing the bacteriophage SP6 promoter, designated pHSTO, permits in vitro transcription of RNAs devoid of vector-derived nucleotides. This vector has been characterized for relative transcriptional activity using constructs which alter the conserved nucleotides extending beyond the SP6 transcriptional initiation site. SP6 polymerase efficiently transcribes cDNA inserts which contain a guanosine (G) nucleotide at position +1 relative to the SP6 promoter; however, inserts with an adenosine (A) or pyrimidine at position +1 are not transcribed. Several cellular and viral cDNAs have been transcribed into translatable messenger RNA using this vector; however, SP6 polymerase will not transcribe the A-T rich untranslated leader from alfalfa mosaic virus RNA 4 efficiently unless the viral mRNA cap site is separated from the transcriptional initiation site by twelve base pairs of vector DNA. Chimeric messenger RNAs were created by linking the untranslated leader sequence of several viral mRNAs to the coding region of barley alpha-amylase, and the resultant mRNAs were translated in a wheat germ extract to determine relative translational efficiencies. The untranslated leader sequences of turnip yellow mosaic virus coat protein mRNA and black beetle virus RNA 2 did not increase translational efficiency, while the tobacco mosaic virus leader stimulated translation significantly. The results indicate that substitution of a cognate untranslated leader sequence with a leader derived from a highly efficient mRNA does not necessarily predict enhanced translational efficiency of the chimeric mRNA.

Different rates of metabolism of two chloroacetanilide herbicides in pioneer 3320 corn.

The in vivo rates of uptake and detoxification of alachlor and metolachlor were determined using Pioneer corn 3320 seedlings. Equal amounts of the radiolabeled herbicides were applied to etiolated coleoptiles and, at various intervals after treatment, the unabsorbed radioactivity was removed and quantified. Analysis of 80% methanol extracts by reverse phase liquid chromatography showed no significant differences in the rate of uptake of metolachlor and alachlor. However, the rate of glutathione conjugation of alachlor in vivo was two- to threefold greater than the rate for metolachlor at 2 and 4 hours after herbicide application. Since the initial step in detoxification is conjugation of the chloroacetanilide to glutathione, the activities of the enzymes responsible for conjugation, the glutathione-S-transferases (GST) were also analyzed in vitro, using crude extracts and the purified GST enzymes. The specific activities of the extracts were consistent with the results in vivo. Using alachlor as a substrate, the specific activity for glutathione conjugation was almost threefold higher than that for metolachlor. Kinetic analysis of purified GST III indicates that the enzyme has a higher affinity for alachlor (K(m)app = 1.69 millimolar) than for metolachlor (K(m)app = 8.9 millimolar).

Evaluation of selectable markers for obtaining stable transformants in the gramineae.

Cell suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) resistance genes. Resistant colonies were obtained at low frequencies (1 x 10(-5) to 2 x 10(-6)) when selected under conditions which were growth inhibitory to protoplasts electroporated without DNA. Southern blot hybridization confirmed stable integration of plasmid DNA into T. monococcum using hygromycin vectors and P. maximum using the methotrexate vector with 1 to 10 copies integrated per haploid genome.

Bacterial expression and isolation of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase.

5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase, EPSPS), an in vivo enzyme target of the herbicide glyphosate (N-phosphonomethyl glycine), was purified from a Petunia hybrida suspension culture line, MP4-G, by a small-scale high-performance chromatographic purification procedure. The cDNA encoding the mature petunia EPSPS (lacking the chloroplast transit sequence) was cloned into a plasmid, pMON342, for expression in Escherichia coli. This clone complemented the EPSPS deficiency of an E. coli aroA- mutant, and the plant enzyme constituted approximately 1% of the total extractable protein. Large-scale purification of the enzyme from E. coli cells resulted in a highly active protein which was homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino terminal sequencing. Antibodies raised against the purified enzyme also reacted with the E. coli EPSPS in Western analyses. The availability of large quantities of the plant enzyme will significantly facilitate mechanistic investigations as well as a comparative study with EPSPS from bacteria and fungi.

Protein trafficking in plant cells.

The cells of higher plants contain distinct subcellular compartments (organelles) that perform specialized functions such as photosynthesis, carbohydrate and lipid metabolism, and so forth. The majority of the protein constituents of plant organelles are formed as cytosolic precursors with N-terminal extensions that direct transport across one or more membrane bilayers in a post- or co-translational fashion. Since the majority of proteins in plant cells are products of nuclear gene expression, there must be precise sorting mechanisms in the cytoplasm that direct proteins to their correct cellular locations. Based on recent studies of protein targeting to chloroplasts and vacuoles, the details of these intracellular sorting mechanisms are becoming clear. The ability to direct proteins to specific compartments within cells provides new opportunities for improvement of plants by genetic manipulation.

5' proximal sequences of a soybean ribulose-1,5-bisphosphate carboxylase small subunit gene direct light and phytochrome controlled transcription.

Two closely related ribulose-1,5-bisphosphate carboxylase small subunit (SSU) genes, SRS1 and SRS4, are transcribed at high levels in soybean plants in response to light. Transgenic petunia plants containing 5' sequences from SRS1 or SRS4 fused to the polypeptide encoding region of a neomycin phosphotransferase (NPTII) gene exhibit selectable kanamycin resistance. Deletion of three ATG codons from the region preceding the normal NPTII translation start site has little effect on the levels of kanamycin resistance in transformed plants. Run-on transcription assays in isolated nuclei demonstrate that transcription of the SRS1/NPTII chimera and the native petunia SSU11A gene subfamily is light regulated and under phytochrome control in leaves of transgenic plants. In young expanding leaves of fully light grown plants, transcription of these genes is markedly reduced within minutes of far-red treatment, while ribosomal DNA and actin gene transcription remains unchanged. This is analogous to the transcriptional response we observed for SRS1 and SRS4 in soybean seedlings. These data suggest (1) that transcription of SSU genes in both soybean and petunia require the continued presence or synthesis of phytochrome in the Pfr form and (2) that 5' sequences are sufficient to direct the phytochrome controlled transcriptional response of the SRS1 gene. In fully expanded mature leaves we found the transcription rates of the native SSU11A gene subfamily, the chimeric SRS1/NPTII gene, the rDNA genes, and several other control genes to be reduced markedly after far-red treatment or after extended periods of darkness. The contrast between results in young and mature leaves is discussed.

Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants.

A chimeric gene encoding the alfalfa mosaic virus (AlMV) coat protein was constructed and introduced into tobacco and tomato plants using Ti plasmid-derived plant transformation vectors. The progeny of the self-fertilized transgenic plants were significantly delayed in symptom development and in some cases completely escaped infection after inoculated with AlMV. The inoculated leaves of the transgenic plants had significantly reduced numbers of lesions and accumulated substantially lower amounts of coat protein due to virus replication than the control plants. These results show that high level expression of the chimeric viral coat protein gene confers protection against AlMV, which differs from other plant viruses in morphology, genome structure, gene expression strategy and early steps in viral replication. Based on our results with AlMV and those reported earlier for tobacco mosaic virus, it appears that genetically engineered cross-protection may be a general method for preventing viral disease in plants.

Virulence of Agrobacterium tumefaciens Strain A281 on Legumes.

This study addresses the basis of host range on legumes of Agrobacterium tumefaciens strain A281, an l,l-succinamopine strain. We tested virulence of T-DNA and vir region constructs from this tumor-inducing (Ti) plasmid with complementary Ti plasmid regions from heterologous nopaline and octopine strains.

Comparison of cauliflower mosaic virus 35S and nopaline synthase promoters in transgenic plants.

We have compared the level of expression of the Cauliflower Mosaic Virus 35S promoter and the nopaline synthase promoter when fused to a common reporter gene. A cassette containing the neomycin phosphotransferase (type II) coding sequence followed by the nopaline synthase 3' nontranslated region was used for transcriptional and translational evaluation of the two different promoters. These chimeric genes were introduced into petunia plants and the copy number of the gene, the steady state level of NPTII transcript and the levels of NPTII enzyme activity were determined. In this paper, we report that the NPT II transcript levels are on the average 30 fold higher in plants containing CaMV 35S promoter and leader sequences than in plants containing the same reporter gene but nopaline synthase promoter and leader sequences. Similarly, plants containing the CaMV 35S promoter had an average of 110 fold higher levels of NPTII enzyme activity than those containing the nopaline synthase promoter. The significance of these results for expression of foreign genes in plants is discussed. In addition, we describe the construction of a convenient plant expression cassette vector (pMON316) which utilizes the CaMV 35S promoter.

Transient expression of electroporated DNA in monocotyledonous and dicotyledonous species.

Transient expression of electroporated DNA was monitored in protoplasts of several monocot and dicot species by assaying for expression of chimeric chloramphenicol acetyltransferase (CAT) gene constructions. Expression was obtained in the dicot species of Daucus carota, Glycine max and Petunia hybrida and the monocot species of Triticum monococcum, Pennisetum purpureum, Panicum maximum, Saccharum officinarum, and a double cross, trispecific hybrid between Pennisetum purpureum, P. americanum, and P. squamulatum. Recovery and viability of protoplasts after electroporation decreased with increasing voltages and capacitance while CAT activity increased up to a critical combination of voltage and capacitance beyond which the activity dramatically decreased. The optimal compromise between DNA uptake and expression versus cell survival was determined for D. carota and applied successfully to the other species. Maximum transient expression occurred 36 hours after electroporation of D. carota. The potential for using this procedure to rapidly assay gene function in dicot and monocot cells and application of this technique to obtain transformed cereals is discussed.

Expression of a soybean ß-conclycinin gene under the control of the Cauliflower Mosaic Virus 35S and 19S promoters in transformed petunia tissues.

A gene encoding the α'-subunit of ß-conglycinin was ligated to the 19S and 35S promoters of Cauliflower Mosaic Virus and introduced into petunia plants on a disarmed Ti-plasmid using Agrobacterium tumefaciens. Transformed cells were regenerated into whole plants and ummunoreactive polypeptides and hybridizable, polyadenylated mRNA were detected in transformed tissues. Expression from the 35S promoter was 10 to 50 times greater than expression from the 19S promoter. The level of immunodetectable polypeptides was greater in seeds than in leaves or callus tissue. In addition, the pattern of α'-polypeptide breakdown products was distinctive in seeds and leaves. We conclude that in seeds the higher levels of the α'-polypeptide reflect enhanced stability of this protein.

The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA.

We used a binary-vector strategy to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. We constructed plasmids (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs in trans with complementary regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results we suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid.