Tomato spotted wilt tospovirus (TSWV) is the type member of the plant-infecting viruses of the genus Tospovirus in the family Bunyaviridae. The three TSWV RNAs are encapsidated with nucleocapsid (N) protein to form ribonucleoprotein (RNP) which serves as the template for viral transcription and replication. Regions of the open reading frame coding for the N protein on the small (S) RNA were subcloned into pET protein expression vectors and expressed in Escherichia coli BL21 (DE3) cells. Full-length N, N amino and carboxy halves, and two N carboxy-terminal regions were expressed and isolated by metal chelate affinity chromatography. The N protein, both of its halves and the extreme carboxy-terminal region, bound cooperatively and irrespective of sequence to radiolabeled single-stranded RNA produced by runoff transcription of clones of either TSWV S RNA or cowpea chlorotic mottle virus RNA3. N protein did not bind to radiolabeled double-stranded TSWV RNA. The density of the synthetic RNase-sensitive N protein-RNA complexes was 1.32 g/ml, similar to the density of authentic Bunyaviridae RNPs. These studies are the first to indicate differences in the nucleic acid binding abilities of Tospovirus and Hantavirus nucleocapsid proteins, the only characterized nucleocapsid proteins of the family Bunyaviridae.

The production of cultivated peanut, an important agronomic crop throughout the United States and the world, is consistently threatened by various diseases and pests. Sclerotinia minor Jagger (S. minor), the causal agent of Sclerotinia blight, is a major threat to peanut production in the Southwestern US, Virginia and North Carolina. Although information on the variability of morphological traits associated with Sclerotinia blight resistance is plentiful, no molecular markers associated with resistance have been reported. The identiWcation of markers would greatly assist peanut geneticists in selecting genotypes to be used in breeding programs. The main objective of this work was to use simple sequence repeat (SSR) primers previously reported for peanut to identify a molecular marker associated with resistance to S. minor. Out of 16 primer pairs used to examine peanut genomic DNA from 39 diVerent genotypes, one pair produced bands at approximately 145 and 100 bp, consistent with either S. minor resistance or susceptibility, respectively. Cloning and sequencing of these bands revealed the region is well conserved among all genotypes tested with the exception of the length of the SSR region, which varies with disease resistance levels. This is the Wrst report of a molecular marker associated with resistance to Sclerotinia blight in peanut. The identiWcation of this marker and development of a PCR-based screening method will prove to be extremely useful to peanut breeders in screening germplasm collections and segregating populations as well as in pyramiding S. minor resistance with other desirable traits into superior peanut lines. Abbreviations ABLAdvanced breeding line AFLP AmpliWed fragment length polymorphism QTL Quantitative trait loci RAPD Random ampliWed polymorphic DNA RFLP Restriction fragment length polymorphism SCAR Sequence characterized ampliWed region SSR Simple sequence repeat Cultivated peanut (Arachis hypogaea L.) is a selfpollinated allotetraploid (2n = 4x = 40), which is

Fungal diseases of peanut are responsible for increased production costs and yield losses of up to 50% for peanut producers in the U.S. Few cultivars with disease resistance have been developed through traditional breeding practices. There is an urgent need for developing cultivated peanut (Arachis hypogaea L.) cultivars that are resistant to the broad spectrum of fungal pathogens that pose a recurring threat to peanut health. Hydrolases such as chitinase and β-1-3-glucanase are known to degrade the cell walls of many fungi that attack plants, making them likely candidates for over-expression through genetic engineering to produce disease-resistant crops. Somatic embryos of the peanut cultivar Okrun were transformed with a chitinase gene from rice, and/or a β-1-3-glucanase from alfalfa via microprojectile bombardment. Regenerated Okrun lines were tested for the presence of the transgenes by polymerase chain reaction (PCR) and Southern blot and for transgene expression by colorimetric assays. Transgenic lines exhibited hydrolase activities 0-37% above levels observed in nontransformed Okrun plants.

No abstract

Isolates of cauliflower mosaic virus (CaMV) differ in host range and symptomatology. Knowledge of their sequence relationships should assist in identifying nucleotide sequences responsible for isolate-specific characters. Complete nucleotide sequences of the DNAs of eight isolates of CaMV were aligned and the aligned sequences were used to analyze phylogenetic relationships by maximum likelihood, bootstrapped parsimony, and distance methods. Isolates found in North America clustered separately from those isolated from other parts of the world. Additional isolates, for which partial sequences were available, were incorporated into phylogenetic analysis of the sequences of genome segments corresponding to individual protein coding regions or the large intergenic region of CaMV DNA. The analysis revealed several instances where the position of an isolate on a tree for one coding region did not agree with the position of the isolate on the tree for the complete genome or with its position on trees for other coding regions. Examination of the distribution of shared residue types of phylogenetically informative positions in anomalous regions suggested that most of the anomalies were due to recombination events during the evolution of the isolates. Application of an algorithm that searches for segments of significant length that are identical between pairs of isolates or contain a significantly high concentration of polymorphisms suggested two additional recombination events between progenitors of the isolates studied and an event between the XinJing isolate and a CaMV not represented in the data set. An earlier phylogenetic origin for CaMV than for carnation etched ring virus, the caulimovirus used as outgroup in these analyses, was deduced from the position of the outgroup with North American isolates in some trees, but with non-North American isolates in other trees.

Peanut (Arachis hypogaea L.) is susceptible to many diseases. In the southwestern USA and other regions where peanut is grown, diseases caused by fungi are a major threat to profitable production. Transgenic peanut lines possessing fungal resistance genes offer an alternative to traditional resistance and fungicide application in managing fungal diseases. Thirty‐two transgenic peanut lines containing antifungal genes (a rice chitinase and/or an alfalfa glucanase) were evaluated for their reaction to Sclerotinia blight caused by Sclerotinia minor Jagger in small field plots (6.1 by 7.6 m) for 3 yr. Peanut lines were arranged in a complete randomized block design with three replications. Disease incidence was recorded throughout the growing season and data were analyzed for statistical significance. Over the 3‐yr period, average disease incidence for the most resistant lines—188, Southwest Runner, 416, 540, and 654—was 0.0, 1.0, 10.0, 14.0, and 16.0%, respectively. The cultivar Okrun was most susceptible with an average disease incidence of 58.0%. All other lines had varying degrees of resistance but averaged at least 15.5% less disease than Okrun over the 3‐yr period. Transgenic peanut lines with partial resistance to Sclerotinia blight were identified which may be useful in traditional breeding programs for fungal resistance.

: Peanut (Arachis hypogaea L.), an economically important crop throughout the world, is susceptible to many types of fungal pathogens. Genetic engineering offers great potential for developing peanut cultivars resistant to a broad spectrum of pathogens that pose a recurring threat to peanut health. The main objective of this research project was to assess the “substantial equivalence” and nutritional safety of biotechnology derived peanut lines. The focus of this study was on genetically modified peanut (GMP) lines developed for growth in the southwestern United States. Three transgenic peanut lines, 188, 540, and 654, and the parent line Okrun were analyzed for their oil, protein, ash, moisture, total dietary fiber, mineral, and fatty acid compositions. Oil content of all the GMP lines was similar to that of the parent line. The GMP line 188 had significantly higher protein content than the parent line, Okrun. Although there were statistical differences in the fatty acid composition among some of the GMP lines and the parent line, the variations were not substantial. The proximate compositions of all peanut lines were within the range reported for traditional peanut varieties. This study indicates that, for the peanut lines studied, genetic modification did not cause substantial unintentional changes in peanut chemical composition, which might reduce the nutritional value of peanut.

In this work, the quantitation of cellular carbohydrates, namely chitin and glucan, in peanut fungal pathogens and baker's yeast was carried out by capillary electrophoresis (CE) and capillary electrochromatography (CEC). The chitin and glucan of the fungi were hydrolyzed by the enzymes chitinase and glucanase, respectively, to their corresponding sugar monomers N-acetylglucosamine (GlcNAc) and glucose (Glc). These two monosaccharides were then tagged with 6-aminoquinoline (6-AQ) to allow their separation and detection in CE and CEC. The 6-AQ derivatives of GlcNAc and Glc formed the basis for the determination by CE and CEC of chitin and glucan in peanut fungi and baker's yeast. Several parameters affecting the separation of the 6-AQ derivatives of GlcNAc and Glc, including the separation voltage and the composition of the running electrolyte, were investigated. Under the optimized separation conditions, the contents of cellular carbohydrates including N-acetylglucosamine, chitin, glucose, and glucan in some fungi, such as Sclerotinia minor, Sclerotium rolfsii, and baker's yeast, were successfully determined. The method described here allowed the assessment of genetic differences in Sclerotium rolfsii isolates from various locations.