Pathogen-induced expression of a blight tolerance transgene in American chestnut.

American chestnut (Castanea dentata) is a susceptible host of the invasive necrotrophic fungus Cryphonectria parasitica, which causes chestnut blight disease. The fungal pathogen attacks chestnut stems by invading wounded tissue and secreting oxalate. This process leads to the death of infected host cells and the formation of cankers, eventually girdling stems and killing the tree above the infections. To reduce damage caused by fungal oxalate, American chestnut has been genetically engineered to express a wheat oxalate oxidase (OxO). This enzyme degrades the oxalate produced by the pathogen and confers elevated tolerance to Cryphonectria parasitica infection. We report new lines of transgenic American chestnut that have been developed with the win3.12 inducible promoter from poplar (Populus deltoides) driving OxO expression. This promoter is responsive to both wounding and pathogen infection, with a low level of baseline expression. Targeted expression of OxO to wounded and infected tissue is sought as an alternative to constitutive expression for potential metabolic resource conservation and transgene stability over the long lifetime of a tree and over successive generations of breeding. Transgenic Castanea dentata lines harbouring the win3.12-OxO construct were evaluated for transgene expression patterns and tolerance to chestnut blight infection. OxO transcript levels were low in uninfected plants, but robust infection-induced expression levels were observed, with one transgenic line reaching levels comparable to those of previously characterized CaMV35S-OxO lines. In chestnut blight infection bioassays, win3.12-OxO lines showed elevated disease tolerance similar to blight-resistant Chinese chestnut (Castanea mollissima) controls.

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.

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.

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.

Chestnut resistance to the blight disease: insights from transcriptome analysis.

BACKGROUND: 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. RESULTS: Over one and a half million cDNA reads were assembled into 34,800 transcript contigs from American chestnut and 48,335 transcript contigs from Chinese chestnut. Chestnut cDNA showed higher coding sequence similarity to genes in other woody plants than in herbaceous species. The number of genes tagged, the length of coding sequences, and the numbers of tagged members within gene families showed that the cDNA dataset provides a good resource for studying the American and Chinese chestnut transcriptomes. In silico analysis of transcript abundance identified hundreds of GDTA in canker versus healthy stem tissues. A significant number of additional DTA genes involved in the defense-response not reported in a previous study were identified here. These DTA genes belong to various pathways involving cell wall biosynthesis, reactive oxygen species (ROS), salicylic acid (SA), ethylene, jasmonic acid (JA), abscissic acid (ABA), and hormone signalling. DTA genes were also identified in the hypersensitive response and programmed cell death (PCD) pathways. These DTA genes are candidates for host resistance to the chestnut blight fungus, Cryphonectria parasitica. CONCLUSIONS: Our data allowed the identification of many genes and gene network candidates for host resistance to the chestnut blight fungus, Cryphonectria parasitica. The similar set of GDTAs in American chestnut and Chinese chestnut suggests that the variation in sensitivity to this pathogen between these species may be the result of different timing and amplitude of the response of the two to the pathogen infection. Resources developed in this study are useful for functional genomics, comparative genomics, resistance breeding and phylogenetics in the Fagaceae.

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.

A randomized controlled trial of household-based flocculant-disinfectant drinking water treatment for diarrhea prevention in rural Guatemala.

We conducted a study to determine if use of a new flocculant-disinfectant home water treatment reduced diarrhea. We randomly assigned 492 rural Guatemalan households to five different water treatment groups: flocculant-disinfectant, flocculant-disinfectant plus a customized vessel, bleach, bleach plus a vessel, and control. During one year of observation, residents of control households had 4.31 episodes of diarrhea per 100 person-weeks, whereas the incidence of diarrhea was 24% lower among residents of households receiving flocculant-disinfectant, 29% lower among those receiving flocculant-disinfectant plus vessel, 25% lower among those receiving bleach, and 12% lower among households receiving bleach plus vessel. In unannounced evaluations of home drinking water, free chlorine was detected in samples from 27% of flocculant-disinfectant households, 35% of flocculant-disinfectant plus vessel households, 35% of bleach households, and 43% of bleach plus vessel households. In a setting where diarrhea was a leading cause of death, intermittent use of home water treatment with flocculant-disinfectant decreased the incidence of diarrhea.