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. 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. 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.

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.

A century ago, Chestnut Blight Disease (CBD) devastated the American chestnut. Backcross breeding has been underway to introgress resistance from Chinese chestnut into surviving American chestnut genotypes. Development of genomic resources for the family Fagaceae, has focused in this project on Castanea mollissima Blume (Chinese chestnut) and Castanea dentata (Marsh.) Borkh (American chestnut) to aid in the backcross breeding effort and in the eventual identification of blight resistance genes through genomic sequencing and map based cloning. A previous study reported partial characterization of the transcriptomes from these two species. Here, further analyses of a larger dataset and assemblies including both 454 and capillary sequences were performed and defense related genes with differential transcript abundance (GDTA) in canker versus healthy stem tissues were identified.

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.