Sclerotinia blight resistance in Virginia-type peanut transformed with a barley oxalate oxidase gene.

Transgenic peanut lines expressing oxalate oxidase, a novel enzyme to peanut, were evaluated for resistance to Sclerotinia blight in naturally infested fields over a 5-year period. Area under the disease progress curve (AUDPC) for transgenic lines in single rows planted with seed from single-plant selections averaged 78, 83, and 90% lower than nontransgenic parents in 2004, 2005, and 2006, respectively. In addition, AUDPC in 14 transgenic lines planted with bulked seed in two-row plots averaged 81% lower compared with nontransgenic parents in 2005 and 86% lower in 16 transgenic lines in 2006. Six transgenic lines yielded 488 to 1,260 kg/ha greater than nontransgenic parents in 2005, and 10 lines yielded 537 to 2,490 kg/ha greater in 2006. Fluazinam (0.58 kg a.i./ha) fungicide sprays in 2008 and 2009 reduced AUDPC in transgenic and nontransgenic lines but AUDPC was lowest in transgenic lines. Without fluazinam, yields of transgenic lines averaged 1,133 to 1,578 kg/ha greater than nontransgenic lines in 2008 and 1,670 to 2,755 kg/ha greater in 2009. These results demonstrated that the insertion of barley oxalate oxidase in peanut conveyed a high level of resistance to Sclerotinia blight, and negated the need for costly fungicide sprays.

A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings.

Phytic acid (myo-inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds. During germination, stored reserves are used as a source of nutrients by the plant seedling. Phytic acid is degraded by the activity of phytases to yield inositol and free phosphate. Due to the lack of phytases in the non-ruminant digestive tract, monogastric animals cannot utilize dietary phytic acid and it is excreted into manure. High phytic acid content in manure results in elevated phosphorus levels in soil and water and accompanying environmental concerns. The use of phytases to degrade seed phytic acid has potential for reducing the negative environmental impact of livestock production. A phytase was purified to electrophoretic homogeneity from cotyledons of germinated soybeans (Glycine max L. Merr.). Peptide sequence data generated from the purified enzyme facilitated the cloning of the phytase sequence (GmPhy) employing a polymerase chain reaction strategy. The introduction of GmPhy into soybean tissue culture resulted in increased phytase activity in transformed cells, which confirmed the identity of the phytase gene. It is surprising that the soybean phytase was unrelated to previously characterized microbial or maize (Zea mays) phytases, which were classified as histidine acid phosphatases. The soybean phytase sequence exhibited a high degree of similarity to purple acid phosphatases, a class of metallophosphoesterases.

Expression of D-myo-inositol-3-phosphate synthase in soybean. Implications for phytic acid biosynthesis.

Phytic acid, a phosphorylated derivative of myo-inositol, functions as the major storage form of phosphorus in plant seeds. Myo-inositol phosphates, including phytic acid, play diverse roles in plants as signal transduction molecules, osmoprotectants, and cell wall constituents. D-myo-inositol-3-phosphate synthase (MIPS EC 5.5.1.4) catalyzes the first step in de novo synthesis of myo-inositol. A soybean (Glycine max) MIPS cDNA (GmMIPS1) was isolated by reverse transcriptase-PCR using consensus primers designed from highly conserved regions in other plant MIPS sequences. Southern-blot analysis and database searches indicated the presence of at least four MIPS genes in the soybean genome. Northern-blot and immunoblot analyses indicated higher MIPS expression and accumulation in immature seeds than in other soybean tissues. MIPS was expressed early in the cotyledonary stage of seed development. The GmMIPS1 expression pattern suggested that it encodes a MIPS isoform that functions in seeds to generate D-myo-inositol-3-phosphate as a substrate for phytic acid biosynthesis.

Soybeans transformed with a fungal phytase gene improve phosphorus availability for broilers.

Male broilers (n = 416) were used to compare the efficacy of providing dietary phytase either as a commercial supplement or as a recombinant protein in transformed soybean. From 7 to 21 d of age, broilers were fed a basal diet containing 0.20% nonphytate P (nP) with additional supplementation by fungal phytase as Natuphos or as raw transformed soybeans expressing recombinant phytase at 400, 800, or 1,200 U/kg. For comparison, broilers were also fed the basal diet containing 0.08, 0.16, or 0.24 added nP. The basal diet was fed as the negative control. Diets were consumed ad libitum as a mash. All excreta were collected from each pen from 18 through 20 d of age, and the birds were killed at 21 d of age. Supplementing the basal diet with nP linearly increased body weight gain, feed efficiency, feed intake, toe ash weight and percentage, and tibia shear force and energy. Phosphorus digestibility decreased linearly as nP level increased, but P excretion increased. Dietary phytase linearly increased growth rate, feed intake, toe ash weight and percentage, tibia shear force and energy, and P digestibility, whereas excretion was decreased. Except for P digestibility, there was no difference in efficacy of responses for performance, bone mineralization, and P excretion between the two sources of phytase. It appears from this study that phytase can improve growth performance of broilers fed low nP diets when provided either as a commercial supplement or in the form of transformed seeds.

Secretion of active recombinant phytase from soybean cell-suspension cultures.

Phytase, an enzyme that degrades the phosphorus storage compound phytate, has the potential to enhance phosphorus availability in animal diets when engineered into soybean (Glycine max) seeds. The phytase gene from Aspergillus niger was inserted into soybean transformation plasmids under control of constitutive and seed-specific promoters, with and without a plant signal sequence. Suspension cultures were used to confirm phytase expression in soybean cells. Phytase mRNA was observed in cultures containing constitutively expressed constructs. Phytase activity was detected in the culture medium from transformants that received constructs containing the plant signal sequence, confirming expectations that the protein would follow the default secretory pathway. Secretion also facilitated characterization of the biochemical properties of recombinant phytase. Soybean-synthesized phytase had a lower molecular mass than did the fungal enzyme. However, deglycosylation of the recombinant and fungal phytase yielded polypeptides of identical molecular mass (49 kD). Temperature and pH optima of the recombinant phytase were indistinguishable from the commercially available fungal phytase. Thermal inactivation studies of the recombinant phytase suggested that the additional protein stability would be required to withstand the elevated temperatures involved in soybean processing.

Responses of Antioxidants to Paraquat in Pea Leaves (Relationships to Resistance).

Differnential sensitivity to the oxidant paraquat was observed in pea (Pisum sativum L.) based on cultivar and leaf age. To assess contributions of inductive responses of the antioxidant enzymes in short-term resistance to oxidative damage, activities of glutathione reductase (GR), superoxide dismutase (SOD), and ascorbate peroxidase (APX) and transcript levels for plastidic GR, Cu,Zn SOD, and cytosolic APX were determined. Responses to paraquat exposure from three different leaf age classes of pea were studied. Resistance was correlated with leaf age, photosynthetic rates, enzyme activities, and pretreatment levels of plastid GR and plastid Cu,Zn SOD transcripts. In response to paraquat, small increases in activities of GR and APX were observed in the more resistant leaves. These changes were not reflected at the mRNA level for the plastidic GR or Cu,Zn SOD. Paraquat-mediated increases in cytosolic APX mRNA occurred in all leaf types, irrespective of resistance. Developmentally controlled mechanisms determining basal antioxidant enzyme activities, and not inductive responses, appear to be critical factors mediating short-term oxidative stress resistance.

Bioproduction of human enzymes in transgenic tobacco.

Transgenic plants have significant potential in the bioproduction of complex human therapeutic proteins due to ease of genetic manipulation, lack of potential contamination with human pathogens, conservation of eukaryotic cell machinery mediating protein modification, and low cost of biomass production. Tobacco has been used as our initial transgenic system because Agrobacterium-mediated transformation is highly efficient, prolific seed production greatly facilitates biomass scale-up, and development of new "health-positive" uses for tobacco has significant regional support. We have targeted bioproduction of complex recombinant human proteins with commercial potential as human pharmaceuticals. Human protein C (hPC), a highly processed serum protease of the coagulation/anticoagulation cascade, was produced at low levels in transgenic tobacco leaves. Analogous to its processing in mammalian systems, tobacco-synthesized hPC appears to undergo multiple proteolytic cleavages, disulfide bond formation, and N-linked glycosylation. Although tobacco-derived hPC has not yet been tested for all posttranslational modifications or for enzymatic (anticlotting) activity, these results are promising and suggest considerable conservation of protein processing machinery between plants and animals. CropTech researchers have also produced the human lysosomal enzyme glucocerebrosidase (hGC) in transgenic tobacco. This glycoprotein has significant commercial potential as replacement therapy in patients with Gaucher's disease. Regular intravenous administration of modified glucocerebrosidase, derived from human placentae or CHO cells, has proven highly effective in reducing disease manifestations in patients with Gaucher's disease. However, the enzyme is expensive (dubbed the "world's most expensive drug" by the media), making it a dramatic model for evaluating the potential of plants to provide a safe, low-cost source of bioactive human enzymes. Transgenic tobacco plants were generated that contained the human glucocerebrosidase cDNA under the control of an inducible plant promoter. hGC expression was demonstrated in plant extracts by enzyme activity assay and immunologic cross-reactivity with anti-hGC antibodies. Tobacco-synthesized hGC comigrates with human placental-derived hGC during electrophoretic separations, is glycosylated, and, most significantly, is enzymatically active. Although expression levels vary depending on transformant and induction protocol, hGC production of > 1 mg/g fresh weight of leaf tissue has been attained in crude extracts. Our studies provide strong support for the utilization of tobacco for high-level production of active hGC for purification and eventual therapeutic use at potentially much reduced costs. Furthermore, this technology should be directly adaptable to the production of a variety of other complex human proteins of biologic and pharmaceutical interest.

Complex organization of the soybean mitochondrial genome: recombination repeats and multiple transcripts at the atpA loci.

Identification of the soybean mitochondrial atpA open reading frame (atpA ORF) was based on sequence similarity with atpA genes in other plant mitochondria and partial protein sequencing. The atpA reading frame ends with four tandem UGA codons which overlap four tandem AUG codons initiating an unidentified reading frame, orf214. The atpA-orf214 region is found in multiple sequence contexts in soybean mitochondrial DNA (mtDNA), which can be attributed to the presence of two recombination repeats. A 1-kb repeat spans 600 nucleotides (nt) of atpA N-terminal coding region and 400 nt of upstream sequence. Its four configurations correspond to two full-length atpA-orf214 genes and two truncated pseudogenes. A 2-kb repeat lies 3 kb downstream from the 1-kb repeat. Restriction maps of cosmid clones suggest that a 10-kb segment containing both repeats is itself duplicated in the mt genome. With two recombination repeats present in a total of three copies per genome, soybean mtDNA is expected to consist of a complex population of subgenomic molecules. Transcription of the atpA loci was analysed by Northern blotting and S1 nuclease protection. The atpA genes express multiple transcripts with one major 3' end and heterogeneous 5' sequences extending several kb upstream of the atpA coding region. The atpA gene and orf214 are co-transcribed on all major transcripts. The pseudogenes do not express stable RNAs.

Cytochrome oxidase subunit II gene is adjacent to an initiator methionine tRNA gene in soybean mitochondrial DNA.

The nucleotide sequence of the cytochrome oxidase subunit II (COII) gene from soybean mitochondria reveals a high degree of sequence conservation to other plant COII sequences. The greatest homology is to pea (97.9%) and the homology extends for some distance both 5' and 3' from the gene. Upstream from the breakpoint in homology with the pea sequence there is a tRNA gene identified as the initiator methionine by homology to known plant tRNA genes. The COII coding sequence appears to be present at a single location in the soybean mitochondrial genome, however some portion of the 5' flanking region is repeated at other sites in the genome. The transcription from the cloned region is complex and is complicated by the presence of the repeated sequence.

Nucleotide sequence of the cytochrome oxidase subunit I gene from soybean mitochondria.

The cytochrome oxidase subunit I gene was isolated from a soybean mitochondrial library and subcloned into M13 for DNA sequencing. The sequences of the gene and flanking regions are presented and compared to the corresponding gene from maize. There is approximately 94% sequence homology between the soybean (dicot) and maize (monocot) coding sequences at the nucleotide level. The soybean sequence exists as a single copy in the mitochondrial genome and contains an open reading frame that could encode a polypeptide of 527 amino acids. There is very little sequence homology between the soybean and maize sequences upstream from the coding regions and none is detected downstream. Even the 3' ends of the COI coding regions differ considerably between soybean and maize. There are many amino acid differences at the carboxy terminus and the predicted polypeptide contains one less amino acid than the maize sequence. Northern analysis of the soybean mitochondrial RNA suggests that this region is actively transcribed and yields two major transcripts.

Nucleotide sequence of the soybean mitochondrial 18S rRNA gene: evidence for a slow rate of divergence in the plant mitochondrial genome.

The nucleotide sequence of the 18S rRNA gene from soybean mitochondria was determined and is presented here in comparison to the 18S rRNA genes from wheat and maize mitochondria. All three genes exhibit remarkable sequence similarity supporting the proposal that there is a slower rate of nucleotide divergence in plant mitochondrial DNA (mtDNA) as compared to the mtDNA of animals. A lower degree of sequence similarity is observed between the dicotyledenous plant soybean and either wheat (84%) or maize (85%) than between the two monocots (96%). A possible secondary structure for the soybean 18S rRNA is presented that is analogous to the proposed structure for the E. coli 16S rRNA.

A novel soybean mitochondrial transcript resulting from a DNA rearrangement involving the 5S rRNA gene.

A tissue culture line of soybean cells (SB-1) is shown to have an unusual arrangement of mitochondrial genes. Fusion of the mitochondrial 5S gene to the distal end of an unidentified gene results in the abundant expression of an 800 nucleotide RNA with the 5S rRNA at its 5' end. Since only the fused 5S rRNA gene is found in this cell line, the functional 5S rRNA must arise by processing of the 800 nucleotide RNA or by intermittent termination of transcription. The 800 nucleotide transcript and its DNA arrangement are not detected at comparable levels in other soybean sources, including the parent plant of the SB-1 tissue culture line. The role of recombination in the origin of this gene fusion and in plant mitochondrial DNA in general is discussed.

Analysis of viral and defective-interfering nucleocapsids in acute and persistent infection by rhabdoviruses.

We have isolated and characterized the RNA of intracellular virus nucleocapsids recovered from a number of cell cultures persistently infected with rabies virus or vesicular stomatitis virus (VSV). VSV persistent infections in BHK21, L cells and Aedes albopictus (mosquito) cells generally showed the presence of large amounts of defective-interfering (DI) nucleocapsid RNA and much smaller amounts of standard (B) nucleocapsid RNA. Persistent infections of BHK21 cells by two rabies virus strains, challenge virus standard (CVS-11) or HEP-Flury, were followed for several months during which time the ratio of DI to B nucleocapsid RNA cycled dramatically. We also observed coordinated fluctuations in the absolute amount of incorporation of [3H]uridine into virus nucleocapsid RNA. Total incorporation was generally highest following a decrease in the relative amount of DI nucleocapsid RNA synthesis. At no time were DI nucleocapsids absent in any of the persistently infected cultures.

Evolution of multiple genome mutations during long-term persistent infection by vesicular stomatitis virus.

Persistent infection of BHK21 cells was established with cloned vesicular somatitis virus plus purified Dl particles and maintained in vitro for over 5 years. After 1 year of persistence, the infectious virus RNA genome had evolved several oligonucleotide map changes, and numerous changes had accumulated by 3.5 years. Additional evolution occurred by the fourth year and continued until the fifth year. In contrast, repeated passage of virus in acute infections of several cell types in vitro or in vivo did not lead to detectable oligonucleotide map changes. The short Dl particle originally used to co-infect with infectious virus in establishing persistent infection has been displaced by an ever present and constantly changing population of other Dl particles of differing sizes and radically differing oligonucleotide maps. We conclude that the genomes of both infectious VSV and its Dl particles undergo continuous evolutionary change during years of persistence. In the infectious virus, these changes involve hundreds of mutations which are usually expressed as poorly replicating, temperature-sensitive, small plaque mutants. These are stable mutants which do not revert to wild-type when passaged repeatedly in acute infections at 37 or 33 degrees C. It appears that the sequestered intracellular environment of persistently infected cells favors rapid and continuous virus evolution.