Bumble bee (Bombus impatiens) survival, pollen usage, and reproduction are not affected by oxalate oxidase at realistic concentrations in American chestnut (Castanea dentata) pollen.

Transgenic American chestnut trees expressing a wheat gene for oxalate oxidase (OxO) can tolerate chestnut blight, but as with any new restoration material, they should be carefully evaluated before being released into the environment. Native pollinators such as bumble bees are of particular interest: Bombus impatiens use pollen for both a source of nutrition and a hive building material. Bees are regular visitors to American chestnut flowers and likely contribute to their pollination, so depending on transgene expression in chestnut pollen, they could be exposed to this novel source of OxO during potential restoration efforts. To evaluate the potential risk to bees from OxO exposure, queenless microcolonies of bumble bees were supplied with American chestnut pollen containing one of two concentrations of OxO, or a no-OxO control. Bees in microcolonies exposed to a conservatively estimated field-realistic concentration of OxO in pollen performed similarly to no-OxO controls; there were no significant differences in survival, bee size, pollen use, hive construction activity, or reproduction. A ten-fold increase in OxO concentration resulted in noticeable but non-significant decreases in some measures of pollen usage and reproduction compared to the no-OxO control. These effects are similar to what is often seen when naturally produced secondary metabolites are supplied to bees at unrealistically high concentrations. Along with the presence of OxO in many other environmental sources, these data collectively suggest that oxalate oxidase at field-realistic concentrations in American chestnut pollen is unlikely to present substantial risk to bumble bees.

Comparative efficacy of gypsy moth (Lepidoptera: Erebidae) entomopathogens on transgenic blight-tolerant and wild-type American, Chinese, and hybrid chestnuts (Fagales: Fagaceae).

American chestnut (Castanea dentata [Marsh.] Borkh.) was once the dominant hardwood species in Eastern North America before an exotic fungal pathogen, Cryphonectria parasitica (Murrill) Barr, functionally eliminated it across its range. One promising approach toward restoring American chestnut to natural forests is development of blight-tolerant trees using genetic transformation. However, transformation and related processes can result in unexpected and unintended phenotypic changes, potentially altering ecological interactions. To assess unintended tritrophic impacts of transgenic American chestnut on plant-herbivore interactions, gypsy moth (Lymantria dispar L.) caterpillars were fed leaf disks excised from two transgenic events, Darling 54 and Darling 58, and four control American chestnut lines. Leaf disks were previously treated with an LD50 dose of either the species-specific Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) or the generalist pathogen Bacillus thuringiensis subsp. kurstaki (Btk). Mortality was quantified and compared to water blank controls. Tree genotype had a strong effect on the efficacies of both pathogens. Larval mortality from Btk-treated foliage from only one transgenic event, Darling 54, differed from its isogenic progenitor, Ellis 1, but was similar to an unrelated wild-type American chestnut control. LdMNPV efficacy was unaffected by genetic transformation. Results suggest that although genetic modification of trees may affect interactions with other nontarget organisms, this may be due to insertion effects, and variation among different genotypes (whether transgenic or wild-type) imparts a greater change in response than transgene presence.

Developing Blight-Tolerant American Chestnut Trees.

An invasive fungal pathogen has reduced the American chestnut (Castanea dentata), once a keystone tree species within its natural range in the eastern United States and Canada, to functional extinction. To help restore this important canopy tree, blight-tolerant American chestnut trees have been developed using an oxalate oxidase-encoding gene from wheat. This enzyme breaks down oxalate, which is produced by the pathogen and forms killing cankers. Expressing oxalate oxidase results in blight tolerance, where the tree and the fungus can coexist, which is a more evolutionarily stable relationship than direct pathogen resistance. Genetic engineering (GE) typically makes a very small change in the tree's genome, potentially avoiding incompatible gene interactions that have been detected in some chestnut hybrids. The GE American chestnut also retains all the wild American chestnut's alleles for habitat adaptation, which are important for a forest ecosystem restoration program.

Transgenic American Chestnuts Do Not Inhibit Germination of Native Seeds or Colonization of Mycorrhizal Fungi.

The American chestnut (Castanea dentata) was once an integral part of eastern United States deciduous forests, with many environmental, economic, and social values. This ended with the introduction of an invasive fungal pathogen that wiped out over three billion trees. Transgenic American chestnuts expressing a gene for oxalate oxidase successfully tolerate infections by this blight fungus, but potential non-target environmental effects should be evaluated before new restoration material is released. Two greenhouse bioassays evaluated belowground interactions between transgenic American chestnuts and neighboring organisms found in their native ecosystems. Potential allelopathy was tested by germinating several types of seeds, all native to American chestnut habitats, in the presence of chestnut leaf litter. Germination was not significantly different in terms of number of seeds germinated or total biomass of germinated seedlings in transgenic and non-transgenic leaf litter. Separately, ectomycorrhizal associations were observed on transgenic and non-transgenic American chestnut roots using field soil inoculum. Root tip colonization was consistently high (>90% colonization) on all plants and not significantly different between any tree types. These observations on mycorrhizal fungi complement previous studies performed on older transgenic lines which expressed oxalate oxidase at lower levels. Along with other environmental impact comparisons, these conclusions provide further evidence that transgenic American chestnuts are not functionally different with regard to ecosystem interactions than non-transgenic American chestnuts.

Chestnut, American (Castanea dentata (Marsh.) Borkh.).

The key to successful transformation of American chestnut is having the correct combination of explant tissue, selectable markers, a very robust DNA delivery system, and a reliable regeneration system. The most important components of this transformation protocol for American chestnut are the following: starting out with rapidly dividing somatic embryos, treating the embryos gently throughout the Agrobacterium inoculation and cocultivation steps, doing the cocultivation step in desiccation plates, and finally transferring the embryos into temporary-immersion bioreactors for selection. None of these departures from standard Agrobacterium transformation protocols is sufficient by itself to achieve transgenic American chestnut, but each component makes a difference, resulting in a highly robust protocol. The average transformation efficiency that can be expected using the described protocol is approximately 170 stable embryogenic transformation events per gram of somatic embryo tissue, a considerable improvement over the 20 transformation events per gram we reported in 2006 (Maynard et al. American chestnut (Castanea dentata (Marsh.) Borkh.) Agrobacterium protocols, 2nd ed., 2006). We have regenerated nearly 100 of these events, containing 23 different gene constructs, into whole plants. As of the fall of 2013, we had a total of 1,275 transgenic chestnut trees planted at eight locations in New York State and one in Virginia. Based on a combination of field-trial inoculations, greenhouse small-stem inoculations, and detached-leaf assays, we have identified three transgenes that produce stronger resistance to chestnut blight than non-transgenic American chestnut. Depending on the transgene and the event, this resistance can be either intermediate between American chestnut and Chinese chestnut, approximately equal to or even higher than the resistance naturally found in Chinese chestnut.

Comparisons of ectomycorrhizal colonization of transgenic american chestnut with those of the wild type, a conventionally bred hybrid, and related fagaceae species.

American chestnut (Castanea dentata [Marsh.] Borkh.) dominated the eastern forests of North America, serving as a keystone species both ecologically and economically until the introduction of the chestnut blight, Cryphonectria parasitica, functionally eradicated the species. Restoration efforts include genetic transformation utilizing genes such as oxalate oxidase to produce potentially blight-resistant chestnut trees that could be released back into the native range. However, before such a release can be undertaken, it is necessary to assess nontarget impacts. Since oxalate oxidase is meant to combat a fungal pathogen, we are particularly interested in potential impacts of this transgene on beneficial fungi. This study compares ectomycorrhizal fungal colonization on a transgenic American chestnut clone expressing enhanced blight resistance to a wild-type American chestnut, a conventionally bred American-Chinese hybrid chestnut, and other Fagaceae species. A greenhouse bioassay used soil from two field sites with different soil types and land use histories. The number of colonized root tips was counted, and fungal species were identified using morphology, restriction fragment length polymorphism (RFLP), and DNA sequencing. Results showed that total ectomycorrhizal colonization varied more by soil type than by tree species. Individual fungal species varied in their colonization rates, but there were no significant differences between colonization on transgenic and wild-type chestnuts. This study shows that the oxalate oxidase gene can increase resistance against Cryphonectria parasitica without changing the colonization rate for ectomycorrhizal species. These findings will be crucial for a potential deregulation of blight-resistant American chestnuts containing the oxalate oxidase gene.

Transgenic American chestnuts show enhanced blight resistance and transmit the trait to T1 progeny.

American chestnut (Castanea dentata) is a classic example of a native keystone species that was nearly eradicated by an introduced fungal pathogen. This report describes progress made toward producing a fully American chestnut tree with enhanced resistance to the blight fungus (Cryphonectria parasitica). The transgenic American chestnut 'Darling4,' produced through an Agrobacterium co-transformation procedure to express a wheat oxalate oxidase gene driven by the VspB vascular promoter, shows enhanced blight resistance at a level intermediate between susceptible American chestnut and resistant Chinese chestnut (Castanea mollissima). Enhanced resistance was identified first with a leaf-inoculation assay using young chestnuts grown indoors, and confirmed with traditional stem inoculations on 3- and 4-year-old field-grown trees. Pollen from 'Darling4' and other events was used to produce transgenic T1 seedlings, which also expressed the enhanced resistance trait in leaf assays. Outcrossed transgenic seedlings have several advantages over tissue-cultured plantlets, including increased genetic diversity and faster initial growth. This represents a major step toward the restoration of the majestic American chestnut.

Chestnut Leaf Inoculation Assay as a Rapid Predictor of Blight Susceptibility.

American chestnuts (Castanea dentata), effectively eliminated from eastern North America by chestnut blight in the twentieth century, are the subject of multiple restoration efforts. Screening individual trees (or tree types) for blight resistance is a critical step in all of these programs. Traditional screening involves inoculating stems of >3-year-old trees with the blight fungus (Cryphonectria parasitica), then measuring resulting cankers a few months later. A quicker, nondestructive, quantitative assay, usable on younger plants, would enhance restoration efforts by speeding the screening process. The assay presented here meets these requirements by inoculating excised leaves with the blight fungus and measuring resulting necrotic lesions. Leaves can be collected from few-month-old seedlings or fully mature trees, and results are measured after less than a week. Leaves from several lines of both American and Chinese chestnuts were inoculated, as well as the congener Allegheny chinquapin, and experimental leaf assay results correlate well with stem assay results from these species. Inoculations with virulent and hypovirulent blight fungi strains also showed relative patterns similar to traditional inoculations. Given the correlations to established stem assay results, this procedure could be a valuable tool to quickly evaluate blight resistance in American chestnut trees used for restoration.

A threshold level of oxalate oxidase transgene expression reduces Cryphonectria parasitica-induced necrosis in a transgenic American chestnut (Castanea dentata) leaf bioassay.

American chestnut (Castanea dentata) was transformed with a wheat oxalate oxidase (oxo) gene in an effort to degrade the oxalic acid (OA) secreted by the fungus Cryphonectria parasitica, thus decreasing its virulence. Expression of OxO was examined under two promoters: a strong constitutive promoter, CaMV 35S, and a predominantly vascular promoter, VspB. Oxo gene transcription was quantified by RT-qPCR. Relative expression of OxO varied approximately 200 fold among events produced with the 35S-OxO. The lowest 35S-OxO event expressed approximately 3,000 fold higher than the highest VspB-OxO event. This was potentially due to the tissue-specific nature of the VspB-controlled expression, the strength of the CaMV 35S constitutive promoter, or position effects. Leaf assays measuring necrotic lesion length were conducted to better understand the relationship between OxO expression level and the blight fungus in planta. A threshold response was observed between the OxO expression level and the C. parasitica lesion length. Five events of the 35S-OxO line showed significantly reduced lesion length compared to the blight-susceptible American chestnut. More importantly, the lesion length in these five events was reduced to the same level as the blight-resistant Chinese chestnut, C. mollissima. This is the first report on enhanced pathogen resistance in transgenic American chestnut.

Optimized GC-MS method to simultaneously quantify acetylated aldose, ketose, and alditol for plant tissues based on derivatization in a methyl sulfoxide/1-methylimidazole system.

The isomers of monosaccharide always produce multiple chromatographic peaks as volatile derivatives during gas chromatography, which may result in the overlapping of different sugar peaks. Whereas reduction and oximation of sugar carbonyl groups for GC analysis do eliminate many isomer derivatives, the approaches create new problems. One ketose can yield two peaks by oximation, and different aldoses and ketoses can yield the same alditol upon reduction, leading to the inability to detect some important monosaccharides. This paper reports an optimal method that yields a single peak per sugar by acetylation directly. By using a methyl sulfoxide (Me2SO)/1-methylimidazole (1-MeIm) system, the carbohydrates in acetic anhydride (Ac2O) esterification reactions were solubilized, and the oxidation that normally occurs was inhibited. The results demonstrate that acetylated derivatives of 23 saccharides had unique peaks, which indicates aldose, ketose, and alditol can be determined simultaneously by GC-MS.

Comparison of the transcriptomes of American chestnut (Castanea dentata) and Chinese chestnut (Castanea mollissima) in response to the chestnut blight infection.

UNLABELLED: BACKGROUND1471-2229-9-51: American chestnut (Castanea dentata) was devastated by an exotic pathogen in the beginning of the twentieth century. This chestnut blight is caused by Cryphonectria parasitica, a fungus that infects stem tissues and kills the trees by girdling them. Because of the great economic and ecological value of this species, significant efforts have been made over the century to combat this disease, but it wasn't until recently that a focused genomics approach was initiated. Prior to the Genomic Tool Development for the Fagaceae project, genomic resources available in public databases for this species were limited to a few hundred ESTs. To identify genes involved in resistance to C. parasitica, we have sequenced the transcriptome from fungal infected and healthy stem tissues collected from blight-sensitive American chestnut and blight-resistant Chinese chestnut (Castanea mollissima) trees using ultra high throughput pyrosequencing. RESULTS: We produced over a million 454 reads, totaling over 250 million bp, from which we generated 40,039 and 28,890 unigenes in total from C. mollissima and C. dentata respectively. The functions of the unigenes, from GO annotation, cover a diverse set of molecular functions and biological processes, among which we identified a large number of genes associated with resistance to stresses and response to biotic stimuli. In silico expression analyses showed that many of the stress response unigenes were expressed more in canker tissues versus healthy stem tissues in both American and Chinese chestnut. Comparative analysis also identified genes belonging to different pathways of plant defense against biotic stresses that are differentially expressed in either American or Chinese chestnut canker tissues. CONCLUSION: Our study resulted in the identification of a large set of cDNA unigenes from American chestnut and Chinese chestnut. The ESTs and unigenes from this study constitute an important resource to the scientific community interested in the discovery of genes involved in various biological processes in Chestnut and other species. The identification of many defense-related genes differentially expressed in canker vs. healthy stem in chestnuts provides many new candidate genes for developing resistance to the chestnut blight and for studying pathways involved in responses of trees to necrotrophic pathogens. We also identified several candidate genes that may underline the difference in resistance to Cryphonectria parasitica between American chestnut and Chinese chestnut.

Transgenic American elm shows reduced Dutch elm disease symptoms and normal mycorrhizal colonization.

The American elm (Ulmus americana L.) was once one of the most common urban trees in eastern North America until Dutch-elm disease (DED), caused by the fungus Ophiostoma novo-ulmi, eliminated most of the mature trees. To enhance DED resistance, Agrobacterium was used to transform American elm with a transgene encoding the synthetic antimicrobial peptide ESF39A, driven by a vascular promoter from American chestnut. Four unique, single-copy transgenic lines were produced and regenerated into whole plants. These lines showed less wilting and significantly less sapwood staining than non-transformed controls after O. novo-ulmi inoculation. Preliminary observations indicated that mycorrhizal colonization was not significantly different between transgenic and wild-type trees. Although the trees tested were too young to ensure stable resistance was achieved, these results indicate that transgenes encoding antimicrobial peptides reduce DED symptoms and therefore hold promise for enhancing pathogen resistance in American elm.

American Elm (Ulmus americana).

American elm (Ulmus americana) is a valuable and sentimental tree species that was decimated by Dutch elm disease in the mid-20th century. Therefore, any methods for modifying American elm or enhancing disease resistance are significant. This protocol describes transformation and tissue culture techniques used on American elm. Leaf pieces containing the midvein and petiole are used for explants. Agrobacterium tumefaciens strain EHA105 is used for transformation, with the binary vector pSE39, containing CaMV35S/nptII as a selectable marker, ACS2/ESF39A as a putative resistance enhancing gene, and CaMV35S/GUS as a reporter.

American Chestnut [Castanea dentata (Marsh.) Borkh].

The key to successful transformation of American chestnut is having the correct combination of explant tissue, selectable and scorable markers, and a reliable regeneration system. Rapidly dividing somatic embryos, growing as proembryogenic masses, are a suitable tissue; the bar gene is a suitable selectable marker in conjunction with 1.0 to 10 mg/L phosphirothricin (PPT); and the mgfp5-ER gene is an effective nondestructive scorable marker. We have also found that the more gently the somatic embryos are treated during the inoculation and co-cultivation steps, the higher the transformation efficiency. The average transformation efficiency that can be expected using the described protocol is approx 20 stable and embryogenic transformation events/g of somatic embryo tissue. Cell line and batch-to-batch deviations both upward and downward should be expected. Finally, somatic embryos can be induced to form shoots, which can then be micropropagated and acclimatized.

Expression of a self-processing, pathogen resistance-enhancing gene construct in Arabidopsis.

A gene cassette, p35S-CNO, was designed to express three gene products driven by a single constitutive CaMV 35S promoter. The individual coding regions were linked in frame to produce a single polyprotein, using spacer sequences encoding a specific heptapeptide cleavage recognition site (ENLYFQS) for the nuclear-inclusion-a (NIa) proteinase of tobacco etch virus (TEV). The protein coding sequences used were: a Trichoderma harzinum endochitinase, a truncated NIa proteinase of TEV, and a wheat oxalate oxidase. When p35S-CNO construct was tested in Arabidopsis thaliana, the polyprotein was properly cleaved after translation and the products exhibited functional enzymatic activity in vivo.

Molecular characterization of a gene encoding a cystatin expressed in the stems of American chestnut (Castanea dentata).

A cDNA clone with similarity to genes encoding cystatin was recently isolated from a cDNA library created using mRNA extracted from stem tissues of Castanea dentata (Marsh.) Borkh. (CASde:Pic1). All of the requisite motifs for inhibitory activity were found upon examination of the deduced amino acid sequence. Reverse transcription-polymerase chain reaction was used to detect the cystatin transcript in healthy stem, leaf and seed tissues, as well as in diseased tissues. Gene fragments encoding this putative cystatin were cloned from American and Chinese (Castanea mollissima Blume) chestnuts and a comparison of these sequences revealed significant differences within the intron, including deletions and alterations in restriction-enzyme sites. The long-term goal of this study is to determine whether the cystatin allele in Chinese chestnut correlates to a resistance gene and, if so, if this allele could be used to enhance resistance in American chestnut.