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

Comparison of the transcriptomes of American chestnut (Castanea dentata) and Chinese chestnut... 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. Page 1 of 11 (page number not for citation purposes) BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 Multiple attempts are being made to develop blight-resist- Background The chestnuts (Castanea), members of the family ant American chestnut genotypes. The search for natural Fagaceae, naturally occur throughout deciduous forests of resistance within American chestnut has been mostly eastern North America, Europe, and Asia [1]. The genus fruitless whereas crosses between American parents exhib- includes ecologically and economically important nut iting limited resistance have produced progeny without and timber producing trees including the Chinese chest- appreciable resistance [2]. The American Chestnut Foun- nut (Castanea mollissima), Japanese chestnut (Castanea cre- dation [11] has been breeding for resistance for over three nata), European Chestnut (Castanea sativa) and American decades by introgression of genes from Chinese chestnut chestnut (Castanea dentata). into American chestnut. However, this approach, although successful in developing blight resistant Ameri- American chestnut was once a dominant tree species in can chestnut varieties, has been slowed by the lack of forest ecosystems of eastern North America, its range genetic tools. Another approach to restoration of chestnut extending from Maine south along the Appalachian is by introduction of hypovirulent genotypes of the path- Mountains to Alabama and westward to the Mississippi ogen, Cryphonectria parasitica [10]. Hypovirulence is a river [2]. In some areas up to 45% of the forest canopy was process in which the virulence of C. parasitica to chestnut comprised of American chestnut [3]. This large, fast-grow- trees is reduced by its infection by fungal viruses. For ing tree played a central role in forest ecosystems, provid- instance, virus-infected individuals of C. parasitica have ing food and habitat for a variety of wildlife. It was also of been shown to produce superficial non-lethal cankers on considerable economic importance, producing strong, European chestnut, and regular treatments with the virus rot-resistant timber, a source of tannins, fuel, wood, and are employed to protect chestnut farms in Europe. How- nuts [4-6]. Because of its utility, rapid growth, ability to ever, attempts to inoculate existing American Chestnut quickly colonize burned or clearcut areas, and edible nuts cankers with hypovirulent strains have met with limited it has been referred to as the "perfect tree" [5]. success and may be impractical for reducing blight symp- toms in the forest due to the large scale of the land mass The reign of the American chestnut came to an abrupt end affected [2]. in the early 1900's when a blight, caused by the fungus, Cryphonectria parasitica, was introduced to North America Development of genomic tools will certainly facilitate the from Asia via infected chestnut nursery stock [2]. The isolation of resistance genes, improve the efficiency of blight was first observed in the Bronx Zoological Park in backcross breeding, and provide genetic reagents for New York in 1904 [7] and within 50 years the American developing resistant varieties by genetic engineering. chestnut was nearly eliminated from the forest [8]. The American Chestnut is transformable using Agrobacterium pathogen infects stem tissues and kills the above ground tumefaciens [12,13] and methods for plant regeneration portions of trees by girdling them. Below ground the trees from somatic embryos have been developed [14-16], per- can survive for many years however, continuously sending mitting the production of many individuals from single up sprouts which are themselves eventually infected. Cry- transformation events. C.A. Maynard's and W.A. Powell's phonectria, which shows a necrotrophic life style is lesser labs have produced transgenic American chestnut trees studied than their biotrophic counterparts. Today, except that are in their second year of field trials (USDA APHIS for occasional trees near the edge of its range which have BRS permit 08-011-105r) demonstrating that all the steps escaped the blight, American chestnut exists primarily as have been developed to genetically engineer this species. shrubs, sprouting from the stumps of blight-topped trees [2,9]. Genomic tools are now being developed to accelerate the identification of resistance genes and the development of Although to a lesser extent, European chestnut (C. sativa) blight resistant American chestnut. In this context, a cen- was also devastated by introduction of C. parasitica [10]. tral objective of The Fagaceae Genomic Tools Project [17] Despite their close relationship, sister species of Castanea is the sequencing of the transcriptomes of chestnut, oak exhibit very different susceptibilities to Cryphonectria and beech species with the long-term goal of isolating infection. Asian chestnuts, the vector for the spread of Cry- genes underlying resistance to the chestnut blight. In this phonectria westward, range from somewhat susceptible to study we used an ultra-high throughput pyrosequencing nearly immune to infection [4]. Most likely, these species approach [18] to quickly generate millions of bases of co-evolved with Cryphonectria. Slow growing cankers are cDNA sequence for plant transcriptome analysis [19-22]. often visible on Chinese and Japanese chestnut trees A comparison of capillary sequencing and next generation although growth and yield of the trees are not substan- sequencing methods [23] showed that pyrosequencing is tially reduced. European chestnut is able to tolerate infec- well adapted for analyzing the transcriptome of both tion slightly more than American chestnut, which has model and non-model species, with lower cost than con- little or no natural resistance to Cryphonectria infection ventional methods such as microarrays, SAGE, or EST [7]. analysis generated using capillary sequencing. Page 2 of 11 (page number not for citation purposes) BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 In total, for all tissues, we have generated and analyzed assembly software (454 Life Sciences) led to the construc- 317,842 and 856,618 sequence reads from American and tion of 7,171 and 14,308 contigs from American and Chi- Chinese chestnut, respectively, for which the Fasta files nese chestnut, respectively (Table 1). Among those can be accessed at the Fagaceae project website [17] and contigs, 247 and 436 were considered large, having an the raw data files in the Short Read Archive at the National average length of 731 nt. There were also 68,860 and Center for Biotechnology Information [24], accession 100,901 sequences from American and Chinese chestnut numbers SRX001799 to SRX001808. Here we focused on cankers, respectively, that did not overlap with other comparing the transcriptomes generated from healthy sequences and were considered as singletons. From the stems and infected canker tissues from American and Chi- canker transcript contigs we were able to tag 5,636 genes nese chestnut. The comparison between the American and from American chestnut and 8,369 from Chinese chest- Chinese chestnut canker transcriptomes enabled us to nut. When those unigenes (transcript contigs) were que- identify a large number of candidate pathogen response ried using BlastX (e-value cutoff: e-10) against the genes for use in studying pathways involved in resistance Cryphonectria parasitica proteome, significant matches to the chestnut blight. were found for 102 (~1.6%) and 213 (~1.5%) of the American and Chinese chestnut unigenes, respectively. Results 454 sequence from Canker tissue libraries 454 sequence from healthy stem tissue libraries The American Chestnut Canker cDNA library was con- Four libraries were constructed from American and Chi- structed from a pool of RNA isolated from canker tissues nese chestnut healthy stem tissues. For Chinese chestnut, of several individuals of one genotype (BA69). The Chi- two separate libraries were constructed from healthy cam- nese chestnut cDNA library was also prepared from RNA bial tissue collected from blight resistant genotypes 'Nan- extracted from several individuals of a single genotype king' and 'Mahogany'. For American chestnut two libraries (Nanking). One plate of sequencing was conducted with were constructed from the genotypes Watertown and Wis- each library using the GS20 model of the 454 system. A niewski. In contrast to the canker transcriptome sequenc- total of 129,508 and 235,635 reads were generated from ing, American chestnut and Chinese chestnut healthy American and Chinese chestnut canker transcriptomes stem transcriptomes were sequenced using the FLX system respectively, in the GS20 runs (NCBI SRA accessions (Roche). A quarter plate of sequencing was conducted for SRX001804 and SRX001799, respectively). The average each American chestnut healthy stem library, while a three length of the reads was 101 nucleotides (nt) (Table 1). The quarter plate worth of sequencing were conducted for the difference in the number of reads generated for the Amer- Chinese chestnut Nanking and Mahogany libraries (Table ican and the Chinese chestnut canker reflects the lower 1). Sequencing of the healthy stem transcriptome from quality of the American chestnut library. In total ~13.3 the two American chestnut genotypes yielded a total of and ~24.0 megabases of cDNA were generated from the 188,334 reads (NCBI SRA accessions SRX001800 and American and Chinese chestnut canker libraries, respec- SRX001801, respectively), with an average read length of tively. Prior to assembly, the canker raw sequence data ~246 nt (Table 1). Slightly more than 2.5 times that from the GS20 was re-analyzed with the improved base- number of reads (488,453) was generated from the two calling software of the new FLX model 454 sequencer. Chinese chestnut healthy stem genotypes (NCBI SRA Contig construction of the 454 reads using the Newbler accessions SRX001805 and SRX001806, respectively) Table 1: Summary of 454 sequencing results obtained in this study for the American and Chinese chestnut transcriptomes Sample System # Plates # of Reads # of bp AL of Reads # Contigs AL of All # Large AL of Large Contigs Contigs Contigs ACCanker GS20 1 129,508 13,080,308 101 7,171 168 247 689 ACHS1 FLX 3/4 126,791 29,828,910 247 11,496 276 885 817 ACHS2 FLX 3/4 162,624 38,165,054 246 9,431 271 691 816 CCCanker GS20 1 235,635 23,799,135 101 14,308 168 436 773 CCMHS FLX 3/4 228,594 56,051,191 246 21,828 344 3,074 851 CCNHS FLX 3/4 259,859 64,271,926 247 28,784 339 4,451 848 Total both 5 2,184,941 428,799,020 198 93.018 261 9,784 799 #, Number; AL, Average length ACCanker, American chestnut genotype BA69 infected stem (canker) cDNA CCCanker, Chinese chestnut variety 'Nanking' infected stem (canker) cDNA CCMHS, Chinese Chestnut variety 'Mahogany' healthy stem cDNA CCNHS, Chinese Chestnut variety 'Nanking' healthy stem cDNA ACHS1, American Chestnut Wisneiwski genotype healthy stem cDNA ACHS2, American Chestnut Watertown genotype healthy stem cDNA Page 3 of 11 (page number not for citation purposes) BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 with an average read length of 247.9 bases (Table 1). In Transcriptome comparison between canker and healthy total, ~46.4 and ~120 megabases of healthy stem tran- stem tissues within Chinese and American chestnut To determine the effect of the infection by the blight caus- scriptome sequence were obtained from American and Chinese chestnut healthy stems, respectively. We gener- ing fungus on gene expression in American and Chinese ated 20,927 contigs for American chestnut healthy stem chestnut trees, we compared the transcriptomes from can- and 50,612 contigs for Chinese chestnut healthy stem kered versus healthy stems (Fig. 2 and Fig. 3). We first using the Newbler assembly software package (454 Life determined how many times a unigene was represented in Sciences) with an average length of 273 nt and 330 nt, each of the libraries based on the number of reads for each respectively. A total of 1,823 and 7,961 contigs from (unigene count). We then determined which genes were American and Chinese chestnut, respectively, were con- in common in the two transcriptomes, versus being spe- sidered large with an average length of 833 nt. This left cific to a library, based on searching the GenBank Acces- 95,483 and 100,779 unassembled singleton reads from sion numbers of the contigs and reads as annotated by American and Chinese chestnut healthy stem sequences, BlastX alignment to the Arabidopsis proteome. Analysis of respectively. From the contigs, a total of 12,883 and canker and healthy stem transcriptomes from American 15,085 genes were tagged from American chestnut and and Chinese chestnut showed that several resistance- Chinese chestnut healthy stem tissues, respectively. related genes were differentially expressed in canker tis- sues (see Additional file 1 and Additional file 2). Those Functional annotation of American and Chinese chestnut genes encode various transcription factors such as WRKY, To determine the possible functions of genes tagged, we zinc finger, Myb, C2 domain, basic helix-loop-helix, used the Gene Ontology (GO) [25] classification system. CCAAT-box, and CCR4-NOT. Several other genes Based on the Arabidopsis proteome, a function could be involved in resistance to biotic stresses were differentially assigned to 83,292 (26%) and 20,391 (28%) of the 454 expressed in canker tissues. Those genes include cin- reads from American and Chinese chestnut respectively. namoyl-CoA reductase (CCR), 4-coumarate–CoA ligase, These percentages are lower than those obtained by hydrolase, kinases, phosphatases, translation factor, ATPases, BLASTx alignments to the Populus proteome (39% and pathogen responsive alpha-dioxygenase, etc (see Additional 46% for American and Chinese chestnut respectively). file 3 and Additional file 4). Some genes such as ABC However, most of the reads (45,804 and 139,230 for transporter, CCAAAT-box, CCR4-NOT and zinc finger seem American and Chinese chestnut respectively) with best to be differentially expressed in canker vs. healthy stem tis- hits to the Populus proteome are for Populus genes that are sues in Chinese chestnut. annotated as having no known function. GO ontology Transcriptome comparison between Chinese and analysis based on the Arabidopsis proteome showed that the distributions of gene functions for cDNA sequences American chestnut canker tissues from American and Chinese chestnut cankers are similar To gain insight into the differences in the response of the (Fig. 1). This expected result indicates that there is no bias American and Chinese chestnut species to infection by the in the construction of the libraries from American and blight-causing agent, we compared the transcriptomes Chinese canker tissues. The functions of genes identified from Chinese chestnut canker tissues and American chest- cover various biological processes. However, hydrolase nut canker tissues (Fig. 1) as described above. This com- and transferase are among the most represented molecu- parison showed that the distribution of gene functions lar function categories. The biological processes most rep- was very similar overall in both the cankers of both spe- resented were transport and protein metabolism. It is cies. However, we observed a small increase in nucleic noteworthy that a larger number of genes involved in acid protein binding and transcription factor molecular response to biotic and abiotic stimuli and stresses were functions in Chinese chestnut cankers. In an opposite pat- identified in Chinese chestnut tissues compared with tern, American chestnut had a small increase in the cate- American chestnut. This difference may be associated with gory "structural molecule activity". We also observed that blight resistance in Chinese chestnut. A similar pattern of the fraction of genes involved in response to stress and GO-annotation function distribution was found when the biotic and abiotic stimuli was slightly higher in American transcriptomes from healthy stem and canker tissues from chestnut canker tissue. However, statistical analysis using American and Chinese chestnut were compared (Fig. 2 GOstat program showed that none of those differences and Fig. 3). As predicted, we observed that the fraction of were statistically significant [26]. Detailed comparison of genes involved in response to stress, biotic and abiotic the transcriptomes showed that many resistance-related stimuli, cell organization and biogenesis processes are genes were differentially expressed in both the American highly represented. The molecular functions most repre- and the Chinese chestnut infection sites (see Additional sented are transferase, protein binding, and hydrolase. file 3 and Additional file 4). Statistical tests as per [27] of the expression data showed that the differential expres- Page 4 of 11 (page number not for citation purposes) BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 AC Canker CC Canker H ch Figure 1 iestnut ca stogram p nker resen tissues a tation of n Ge d biological ne Onto processe logy classification of putative molecular s in which they are involved function of unigenes from American and Chinese Histogram presentation of Gene Ontology classification of putative molecular function of unigenes from American and Chinese chestnut canker tissues and biological processes in which they are involved. sion of many of the resistance-related genes was statisti- used to analyze the transcriptome in non model plants. cally significant. Examples of the resistance-related genes However, deep EST sequencing using capillary sequenc- preferentially expressed in American chestnut (based on ing, which requires cDNA cloning and individual DNA the difference in reads per unigene) include genes encod- preparations for each clone, is time consuming and very ing proteins such as SNF7, laccase, CCR, cinnamyl alcohol costly. Bead-based pyrosequencing introduced recently dehydrogenase (CAD), expansin, F-box proteins, FAD- [18] constitutes a better alternative for transcriptomics. binding protein, proteins named disease-resistance- The high number of reads generated per run together with responsive, etc. Most of those genes play an important the low sequencing error rate in the contigs obtained role in plant response to pathogen infection. Genes pre- makes it a good tool to deeply sequence the transcriptome senting relatively high expression in Chinese chestnut of plants. This approach has been used successfully for encode proteins such as mitogen-activated kinase, Myb analyzing the transcriptomes of maize and Arabidopsis transcription factors, pathogen-responsive alpha-dioxyge- [19-22] and we have applied it to the non-model tree spe- nase, laccase, cytochrome P450, F-box proteins, SNF7, cies Castanea dentata and C. mollissima. CCR, succinyl-CoA ligase, etc. However, most of the gene expression differences in Chinese chestnut were not statis- Before this project, only a few hundred chestnut tically significant in our data set. sequences had been deposited in the EST database (dbEST) at NCBI. The data presented here represent the first large effort by the Fagaceae Genomic Tools Develop- Discussion American and Chinese chestnut transcriptome sequencing ment project to generate cDNA resources and analyze the Advances in DNA sequencing technology during the last transcriptomes of American and Chinese chestnut. These decade have dramatically impacted genome sequencing resources are public and the sequences can be accessed in and transcriptome analysis. Techniques such as microar- a searchable database at the project website [17], or as raw rays and SAGE have facilitated transcriptome analysis at sequence data at the NCBI Short Read Archive (accession large scale from numerous plants. However, those tech- above). In total, our study generated 171 Mb and 78 Mb niques could be used only for model plants with known and tagged 40,039 and 28,890 genes from Chinese chest- genome sequences. EST sequencing has been successfully nut and American chestnut, respectively. A fraction (rang- Page 5 of 11 (page number not for citation purposes) cellular component unknown other membranes other cellular components other intracellular components chloroplast mitochondria other cytoplasmic components nucleus ribosome plastid cytosol plasma membrane cell wall golgi apparatus ER extracellular DNA or RNA binding hydrolase activity kinase activity nucleic acid binding nucleotide binding other binding other enzyme activity other molecular function protein binding structural molecule activity transcription factor activity transferase activity transporter activity unknown molecular function receptor binding or activity cell organization and biogenesis developmental processes DNA or RNA metabolism electron transport or energy pathways other biological processes other cellular processes other metabolic processes protein metabolism response to abiotic or biotic stimulus response to stress signal transduction transcription transport unknown biological processes BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 AC Canker AC Healthy stem H h Figure 2 eistogram presentation of Gen althy stem and canker tissues and biolog e Ontology classifica ical procetion of putative molecular functions sses in which they are involved of unigenes from American chestnut Histogram presentation of Gene Ontology classification of putative molecular functions of unigenes from American chestnut healthy stem and canker tissues and biological processes in which they are involved. ing between 14% and 21%) of American and Chinese also include a large number of genes known to be chestnut unigenes that could not be annotated using the involved in response to biotic and abiotic stimuli and Arabidopsis proteome could however be annotated using stress in general. These gene sequences constitute a very the Populus proteome. Most of the genes with no hits to important resource to the scientific community working the Arabidopsis proteome encoded proteins annotated on chestnut blight resistance as well as those interested in with unknown functions in the poplar genome, however. gene discovery in Fagaceae species. Those genes could correspond to either tree specific genes or sequences that have diverged in Populus and chestnut By taking into consideration only the sequences that have beyond recognizable homology to Arabidopsis using the homologies in the Arabidopsis proteome, two plates of 454 Blast algorithm. Moreover, over 50% of the 454 reads sequences from American chestnut and Chinese chestnut could not be annotated using either the Arabidopsis pro- were enough to generate ~13,000 and ~15,000 unigenes teome or the Populus proteome. A query against the Fungi from each species. This number represents 52% and 60% database at NCBI excluded a Cryphonectria origin for a of American and Chinese chestnut transcriptome respec- small fraction of those reads (~3% for both species). tively, assuming that the two chestnut species have a sim- While a fraction of the remaining sequences may corre- ilar gene number as Arabidopsis. Such breadth and depth spond to 3' or 5' untranslated regions, non coding RNAs, (the number of reads per gene varying between 60 and or short sequences not containing a known protein 178) of coverage by 454 gene tagging, makes this tech- domain, a large number may correspond to potential nique a good tool for quantifying the expression level of Chestnut-specific genes. A similar situation was found sets of genes involved in various developmental stages or when analyzing the transcription of Eschscholzia califor- physiological conditions. cDNA sequences generated nica, Persea americana, and Aristolochia fimbriata [28] and from both species cover various biological processes and (Kerr Wall, personal communication). The two sets of molecular functions indicating that 454 sequencing con- unigenes from Chinese chestnut and American chestnut stitutes a powerful tool for sequencing the transcriptome Page 6 of 11 (page number not for citation purposes) cellular component unknown other membranes other cellular components other intracellular components chloroplast mitochondria other cytoplasmic components nucleus ribosome plastid cytosol plasma membrane cell wall golgi apparatus ER extracellular DNA or RNA binding hydrolase activity kinase activity nucleic acid binding nucleotide binding other binding other enzyme activity other molecular function protein binding structural molecule activity transcription factor activity transferase activity transporter activity unknown molecular function receptor binding or activity cell organization and biogenesis developmental processes DNA or RNA metabolism electron transport or energy pathways other biological processes other cellular processes other metabolic processes protein metabolism response to abiotic or biotic stimulus response to stress signal transduction transcription transport unknown biological processes BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 CC Canker CC Healthy stem h Figure 3 Heistogram presentation of Gen althy stem and canker tissues and biolog e Ontology classificati ical processe on of putative molecula s in which they are invo r functions lved of unigenes from Chinese chestnut Histogram presentation of Gene Ontology classification of putative molecular functions of unigenes from Chi- nese chestnut healthy stem and canker tissues and biological processes in which they are involved. of non model species. These results confirm that pyrose- members (unigenes or reads). Two members of the hydro- quencing constitutes a powerful tool for transcriptome lase group are the glycosyl hydrolase family 3 proteins, characterization and gene discovery. each of which was found five times in our transcriptome data. Glycosyl hydrolases break the bonds between carbo- Transcriptome comparison between canker tissues from hydrates and are involved in expansion and degradation Castanea mollissima and Castanea dentata of cell walls [30]. It is possible that some of the genes with GO annotation analyses showed that, overall, canker tis- hydrolase activity identified in Chinese chestnut canker sues from both species present a similar transcriptome. tissue are contaminants from the pathogen fungi (Cryph- Gene function categories associated with metabolic proc- onectria) mycelium within the canker and function by ess are highly represented in both transcriptomes. The cat- weakening the plant cell wall to facilitate fungal entry. egory represented the most is composed of genes However, analysis of these hydrolase sequences showed associated with various metabolic processes as previously that they are more similar to other plant hydrolase genes described in other systems such as cassava [29]. The sec- than to fungal hydrolase sequences. This suggests that ond most highly represented category includes genes these hydrolase proteins are of plant origin and function involved in resistance to stress and response to biotic and either by strengthening cell walls against pathogen entry abiotic stimuli. Detailed analysis of the 454 sequences or in the programmed cell death response of the cells at from both Chinese and American chestnut showed that the fungal infection site in the chestnut stem. A second the tagged genes included a large number associated with functional category well represented is kinase activity. resistance to biotic and abiotic stresses. These include Such genes are involved in signaling in pathogen infection genes involved in pathogen recognition and signaling, and play a key role in plant defense response. A third func- transcription factors, and resistance genes. Comparison of tional category observed in the chestnut transcriptomes is genes highly expressed in the canker tissues of both Amer- represented by transcription factors or genes associated ican and Chinese chestnut showed that a fraction were with RNA or protein binding. Such genes may modulate either preferentially expressed in American chestnut or in the expression of resistance genes in response to the path- Chinese chestnut. Genes with hydrolase activity repre- ogen infection. sented the functional category with the largest number of Page 7 of 11 (page number not for citation purposes) Relative number of genes, % cellular component unknown other membranes other cellular components other intracellular components chloroplast mitochondria other cytoplasmic components nucleus ribosome plastid cytosol plasma membrane cell wall golgi apparatus ER extracellular DNA or RNA binding hydrolase activity kinase activity nucleic acid binding nucleotide binding other binding other enzyme activity other molecular function protein binding structural molecule activity transcription factor activity transferase activity transporter activity unknown molecular function receptor binding or activity cell organization and biogenesis developmental processes DNA or RNA metabolism electron transport or energy pathways other biological processes other cellular processes other metabolic processes protein metabolism response to abiotic or biotic stimulus response to stress signal transduction transcription transport unknown biological processes BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 Candidate genes involved in chestnut response to the gene PAL2, a key enzyme in phenylpropanoid and Cryphonectria parasitica infection lignin biosynthesis [51]. WRKY transcription factors have Among genes that were found to be differentially been shown to fine tune the response of plants to chal- expressed in American or Chinese chestnut or both, sev- lenge with pathogens [52]. SNF genes interact with other eral are known to be involved in various processes of plant genes, such as SnRK1, which regulate glucose metabolism, defense against pathogens such as cell death related to cell defense and other cellular processes. hypersensitivity response, construction of a physical bar- rier to block the pathogen progression, as well as systemic Overall, this study allowed us to conclude that chestnut resistance. Among genes involved in hypersensitivity cell trees respond to Cryphonectria parasitica infection by acti- death, we found ABC transporter, C2-domain-containing vating both local and systemic resistance responses. The gene, methylenetetrahydrofolate reductase, elongation factor-1 trees try first to block the progression of the pathogen by alpha, and peroxidase. Such genes are involved in control- increasing the expression of hydrolases, lignin synthesis, ling the extent of the cell death in the defense response and cell death. The infection is also sensed by mitogen [31-34]. Pleiotropic drug resistance genes (ABC trans- kinases, which activate other transcriptions factors such as porter family), which are involved in jasmonic acid path- AP2, Myb, and WRKY, which in turn induce the expression way response, induce the secretion of secondary of genes from the phenylpropanoid, jasmonic acid, oligo- metabolites such as diterpenes that inhibit the growth of chitosan, and other pathways that are involved in resist- invading organisms [35-37]. The other category of genes ance to pathogens [53,54]. that seems to be involved in plant resistance to the patho- gen encodes proteins involved in lignin biosynthesis such Conclusion as CCR, CAD, o-methyltransferase 1, cytochrome P450, 4- In conclusion, this study allowed us to (i) Obtain over coumarate–CoA ligase, succinyl-CoA ligase, S-adenosyl- 28,000 and 40,000 unigenes from American and Chinese methionine synthase 3, and S-adenosylmethionine syn- chestnut, (ii) Compare the transcriptomes of American thase 2. Previous studies [38-42] showed that genes and Chinese chestnut following infection by Cryphonectria involved in lignin synthesis are over-expressed in various parasitica, (iii) Identify potential pathways involved in plants when they were challenged with pathogens. chestnut resistance to the Cryphonectria parasitica, and (iv) Among other resistance genes over-expressed in American Identify several candidate genes for resistance to necro- and Chinese chestnut, we found several laccase genes, trophic fungal pathogens in trees. which also belong to the phenylpropanoid pathway. Polyphenol oxidases (PPO) catalyzing the oxygen- Methods American and Chinese chestnut materials dependent oxidation of phenols to quinines, have been demonstrated to increase tomato plant resistance against Healthy cambial tissue was collected from the American Pseudomonas syringae [43]. We also found several ATP- chestnut genotypes 'Watertown' and "Wisniewski' grow- binding cassette transport proteins, which are involved in ing at the Connecticut Agricultural Experiment Station, both constitutive and jasmonic acid-dependent induced Lockwood Farm, Hamden CT. Canker tissue was collected defense [35]. Chestnut plants seem also to activate the from the American chestnut genotype BA69 growing at expression of genes involved in systemic resistance when The American Chestnut Foundation Meadowview they are challenged with the blight fungus. Among genes Research Farm, Meadowview VA. Healthy cambial tissue belonging to this pathway, we identified omega-3 fatty acid was collected from the Chinese chestnut blight resistant desaturase, suppressor of fatty acid desaturase deficiency (SFD1 genotypes 'Nanking' and 'Mahogany' growing at The and SFD2), Ras-related GTP-binding which are required for American Chestnut Foundation Meadowview Research systemic resistance [44,45]. ATPase was found to be over- Farm, Meadowview VA. Canker tissues were collected expressed in American and Chinese chestnut. This gene is from Chinese chestnut genotype 'Nanking' growing at required for the attenuation of the hypersensitive Meadowview Research Farm. To create cankers, the stems response [43]. Among genes involved in signaling, we of Chestnut trees were inoculated with the hypervirulent found several genes such as mitogen activated protein. This Cryphonectria strain EP155 as described by Hebard and protein kinase activates both local resistance and basal collaborators [55]. Canker tissues were sampled 5 and 14 resistance [38,46,47]. It also appears from our data that days post-inoculation and pooled before RNA prepara- metabolic flux may be involved in the chestnut resistance tion. All samples were collected in liquid nitrogen and fro- to the fungus. Several other genes involved in the regula- zen at -80°C until use. tion of resistance gene expression such as Acetyl co-enzyme A carboxyltransferase (CAC3), SNF, and several transcrip- RNA preparation and cDNA library synthesis tion factors such as WRKY, Zinc finger, Myb, etc were iden- Total RNA was prepared by the method of Chang and col- tified. Myb genes are involved in regulation of disease laborators [56]. Three to five grams of frozen tissue were resistance genes [48-50]; they regulate the expression of weighed, ground to a fine powder under liquid nitrogen, Page 8 of 11 (page number not for citation purposes) BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 and dispersed in CTAB buffer. Following 2 chloroform nut canker and Chinese chestnut canker tissues as well as extractions, RNA was precipitated with LiCl , again between canker and healthy stem tissues within each spe- extracted with chloroform and precipitated with ethanol. cies was done using test developed by Dr. Claverie's team The resulting RNA pellet was resuspended in 40–100 μl of [27]. DEPC-treated water, and the quality was assessed with an Agilent Technologies 2100 Bioanalyzer (Agilent Technol- Abbreviations ogies). Poly(A) RNA was then separated from total RNA CAD: cinnamyl alcohol dehydrogenase; CCR: cinnamyl- using the Poly(A) Purist kit (Ambion) and the quality CoA reductase; cDNA: complementary DNA; EST: assessed with an Agilent Technologies 2100 Bioanalyzer expressed sequence tag; GO: Gene Ontology; Mb: mega- (Agilent Technologies). cDNA was synthesized from the bases; NCBI: National Center for Biotechnology Informa- mRNA using the Just cDNA kit (Stratagene) using random tion; nt: nucleotide; SAGE: Serial Analysis of Gene hexamer primers provided with the kit to obtain better 5' Expression. to 3' coverage of transcripts than is possible using Poly(A) priming alone. Authors' contributions AB contributed to extracting RNA and making the 454 The resulting cDNA was used to construct a 454 library libraries, curated and analyzed the data, supervised the following the supplier's instructions (Roche Diagnostics). work SC and YZ, and wrote the paper. CS and YZ contrib- The sequencing was conducted at Penn State University uted to the bioinformatics analyses. DSD collected tissue using an FLX model 454 DNA sequencer (Roche Diagnos- samples, prepared RNA, cDNA and 454 libraries, and tics). helped prepare the first draft of the manuscript. KB and WAP contributed to the RNA preparation and discussion 454 library construction and sequencing of disease response gene candidates. NW manages all 454 libraries were constructed as described previously aspects of the Genomic Tool Development for the [57]. In summary, cDNAs were sheared by nebulization to Fagaceae Project. RS is the Principle Investigator of the yield fragments approximately 500 bp in length. Adaptor Genomic Tool Development for the Fagaceae Project and sequences were ligated to fragmented cDNA, which were was responsible for oversight, budget, obtaining the fund- subsequently immobilized on beads. The DNA fragments ing for the project, and contributing advice at each step of were then denatured to yield a single stranded DNA the research. This work was conducted in the laboratory of library which was amplified by emulsion PCR for JC, who initiated the research with American Chestnut sequencing. Sequencing of the library was performed on a Foundation funding, co-directs the 454 sequencing facil- GS20 and an FLX model 454 DNA sequencer (454 Life ity at Penn State, and contributed to the development of Sciences). All raw 454 sequence data generated in this 454 sequencing protocols, evaluation and discussion of study is available at the Short Read Archive at the National the results, and preparation of the manuscript. Center for Biotechnology Information [24], specifically NCBI accession numbers SRX001799, SRX001800, Additional material SRX001801, SRX001804, RX001805, and SRX001806 (submission SRP000395). Additional File 1 Genes more highly expressed in canker tissues than healthy stem tis- Transcript Assembly and analysis sues of American Chestnut. The data from the 454 read sequences were assembled Click here for file into transcript contigs using Newbler Assembler software [http://www.biomedcentral.com/content/supplementary/1471- 2229-9-51-S1.docx] (Roche). Reads from each library were assembled sepa- rately. The unigene (contigs and remaining unique single- Additional File 2 tons) sequences were annotated by query against the Genes more highly expressed in canker tissues than in healthy stem tis- proteomes of Arabidopsis [58]) and Populus [59] and the sues of Chinese Chestnut. predicted proteome for the blight fungus Cryphonectria Click here for file parasitica [60] using Blastx (e-value cutoff of -10). The [http://www.biomedcentral.com/content/supplementary/1471- Gene Ontology (GO) (Consortium, 2008) system was 2229-9-51-S2.docx] used to summarize possible functional classifications of Additional File 3 the unigenes via assignment of Arabidopsis gene identifiers Disease and Defense Response Genes More Highly Expressed in with the strongest BLASTx alignments to the correspond- Infected Tissues of Chinese Chestnut (CC) than in American Chestnut ing chestnut 454 reads. Comparison of the distribution of (AC). biological processes or molecular function obtained using Click here for file Go annotation was done using GOstat program [26]. [http://www.biomedcentral.com/content/supplementary/1471- Comparison of gene expression between American chest- 2229-9-51-S3.doc] Page 9 of 11 (page number not for citation purposes) BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 15. Carraway DT, Merkle SA: Plantlet regeneration fomr somatic embryos of American Chestnut. Can J For Res 1997, Additional File 4 27:1805-1812. Disease- and Defense- Response Genes More Highly Expressed in 16. Xing Z, Powell WA, Maynard CA: Development and germina- Infected Tissues of American Chestnut (AC) than in Chinese Chestnut tion of American chestnut somatic embryos. Plant Cell, Tissue and Organ Culture 1999, 57:47-55. (CC). 17. The Fagaceae Genomic Tools Project [http:// Click here for file www.fagaceae.org] [http://www.biomedcentral.com/content/supplementary/1471- 18. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, 2229-9-51-S4.doc] Berka J, Braverman MS, Chen YJ, Chen Z, et al.: Genome sequenc- ing in microfabricated high-density picolitre reactors. Nature 2005, 437(7057):376-380. 19. Weber AP, Weber KL, Carr K, Wilkerson C, Ohlrogge JB: Sampling the Arabidopsis transcriptome with massively parallel pyro- Acknowledgements sequencing. Plant Physiol 2007, 144(1):32-42. 20. Redestig H, Weicht D, Selbig J, Hannah MA: Transcription factor We would like to thank Dr. Sandra L. Anagnostakis and Dr. Fredrick V. target prediction using multiple short expression time series Hebard for providing us with chestnut tissues. We also thank Dr. Haiying from Arabidopsis thaliana. BMC Bioinformatics 2007, 8:454. Liang for her help with cDNA library preparation as well as our colleagues 21. Ohtsu K, Smith MB, Emrich SJ, Borsuk LA, Zhou R, Chen T, Zhang X, Dr Stephan Schuster, Lynn Tomsho, and Michael Packard for 454 library Timmermans MC, Beck J, Buckner B, et al.: Global gene expression preparation and for expert technical assistance with 454 sequencing. We analysis of the shoot apical meristem of maize (Zea mays L.). Plant J 2007, 52(3):391-404. thank Kerr Wall, Alex Choi and Urmila Plakkat for their help with sequence 22. Emrich SJ, Barbazuk WB, Li L, Schnable PS: Gene discovery and analysis, tables and figure preparation. 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Plant Mol Biol 2004, 55(4):501-520. available free of charge to the entire biomedical community 55. Hebard F, Griffin G, Elkins J: Developmental histopathology of cankers incited by virulent and hypovirulent Endothia para- peer reviewed and published immediately upon acceptance sitica on susceptible and resistant chestnut trees. Phytopathol- cited in PubMed and archived on PubMed Central ogy 1984, 74:140-149. 56. Chang S, Puryear J, Cairney J: A simple and efficient method for yours — you keep the copyright isolating RNA from pine trees. Plant Mol Biol Rep 1993, BioMedcentral 11:113-116. Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 11 of 11 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Plant Biology Springer Journals

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

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Copyright © 2009 by Barakat et al; licensee BioMed Central Ltd.
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Life Sciences; Plant Sciences; Agriculture; Tree Biology
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Abstract

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. Page 1 of 11 (page number not for citation purposes) BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 Multiple attempts are being made to develop blight-resist- Background The chestnuts (Castanea), members of the family ant American chestnut genotypes. The search for natural Fagaceae, naturally occur throughout deciduous forests of resistance within American chestnut has been mostly eastern North America, Europe, and Asia [1]. The genus fruitless whereas crosses between American parents exhib- includes ecologically and economically important nut iting limited resistance have produced progeny without and timber producing trees including the Chinese chest- appreciable resistance [2]. The American Chestnut Foun- nut (Castanea mollissima), Japanese chestnut (Castanea cre- dation [11] has been breeding for resistance for over three nata), European Chestnut (Castanea sativa) and American decades by introgression of genes from Chinese chestnut chestnut (Castanea dentata). into American chestnut. However, this approach, although successful in developing blight resistant Ameri- American chestnut was once a dominant tree species in can chestnut varieties, has been slowed by the lack of forest ecosystems of eastern North America, its range genetic tools. Another approach to restoration of chestnut extending from Maine south along the Appalachian is by introduction of hypovirulent genotypes of the path- Mountains to Alabama and westward to the Mississippi ogen, Cryphonectria parasitica [10]. Hypovirulence is a river [2]. In some areas up to 45% of the forest canopy was process in which the virulence of C. parasitica to chestnut comprised of American chestnut [3]. This large, fast-grow- trees is reduced by its infection by fungal viruses. For ing tree played a central role in forest ecosystems, provid- instance, virus-infected individuals of C. parasitica have ing food and habitat for a variety of wildlife. It was also of been shown to produce superficial non-lethal cankers on considerable economic importance, producing strong, European chestnut, and regular treatments with the virus rot-resistant timber, a source of tannins, fuel, wood, and are employed to protect chestnut farms in Europe. How- nuts [4-6]. Because of its utility, rapid growth, ability to ever, attempts to inoculate existing American Chestnut quickly colonize burned or clearcut areas, and edible nuts cankers with hypovirulent strains have met with limited it has been referred to as the "perfect tree" [5]. success and may be impractical for reducing blight symp- toms in the forest due to the large scale of the land mass The reign of the American chestnut came to an abrupt end affected [2]. in the early 1900's when a blight, caused by the fungus, Cryphonectria parasitica, was introduced to North America Development of genomic tools will certainly facilitate the from Asia via infected chestnut nursery stock [2]. The isolation of resistance genes, improve the efficiency of blight was first observed in the Bronx Zoological Park in backcross breeding, and provide genetic reagents for New York in 1904 [7] and within 50 years the American developing resistant varieties by genetic engineering. chestnut was nearly eliminated from the forest [8]. The American Chestnut is transformable using Agrobacterium pathogen infects stem tissues and kills the above ground tumefaciens [12,13] and methods for plant regeneration portions of trees by girdling them. Below ground the trees from somatic embryos have been developed [14-16], per- can survive for many years however, continuously sending mitting the production of many individuals from single up sprouts which are themselves eventually infected. Cry- transformation events. C.A. Maynard's and W.A. Powell's phonectria, which shows a necrotrophic life style is lesser labs have produced transgenic American chestnut trees studied than their biotrophic counterparts. Today, except that are in their second year of field trials (USDA APHIS for occasional trees near the edge of its range which have BRS permit 08-011-105r) demonstrating that all the steps escaped the blight, American chestnut exists primarily as have been developed to genetically engineer this species. shrubs, sprouting from the stumps of blight-topped trees [2,9]. Genomic tools are now being developed to accelerate the identification of resistance genes and the development of Although to a lesser extent, European chestnut (C. sativa) blight resistant American chestnut. In this context, a cen- was also devastated by introduction of C. parasitica [10]. tral objective of The Fagaceae Genomic Tools Project [17] Despite their close relationship, sister species of Castanea is the sequencing of the transcriptomes of chestnut, oak exhibit very different susceptibilities to Cryphonectria and beech species with the long-term goal of isolating infection. Asian chestnuts, the vector for the spread of Cry- genes underlying resistance to the chestnut blight. In this phonectria westward, range from somewhat susceptible to study we used an ultra-high throughput pyrosequencing nearly immune to infection [4]. Most likely, these species approach [18] to quickly generate millions of bases of co-evolved with Cryphonectria. Slow growing cankers are cDNA sequence for plant transcriptome analysis [19-22]. often visible on Chinese and Japanese chestnut trees A comparison of capillary sequencing and next generation although growth and yield of the trees are not substan- sequencing methods [23] showed that pyrosequencing is tially reduced. European chestnut is able to tolerate infec- well adapted for analyzing the transcriptome of both tion slightly more than American chestnut, which has model and non-model species, with lower cost than con- little or no natural resistance to Cryphonectria infection ventional methods such as microarrays, SAGE, or EST [7]. analysis generated using capillary sequencing. Page 2 of 11 (page number not for citation purposes) BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 In total, for all tissues, we have generated and analyzed assembly software (454 Life Sciences) led to the construc- 317,842 and 856,618 sequence reads from American and tion of 7,171 and 14,308 contigs from American and Chi- Chinese chestnut, respectively, for which the Fasta files nese chestnut, respectively (Table 1). Among those can be accessed at the Fagaceae project website [17] and contigs, 247 and 436 were considered large, having an the raw data files in the Short Read Archive at the National average length of 731 nt. There were also 68,860 and Center for Biotechnology Information [24], accession 100,901 sequences from American and Chinese chestnut numbers SRX001799 to SRX001808. Here we focused on cankers, respectively, that did not overlap with other comparing the transcriptomes generated from healthy sequences and were considered as singletons. From the stems and infected canker tissues from American and Chi- canker transcript contigs we were able to tag 5,636 genes nese chestnut. The comparison between the American and from American chestnut and 8,369 from Chinese chest- Chinese chestnut canker transcriptomes enabled us to nut. When those unigenes (transcript contigs) were que- identify a large number of candidate pathogen response ried using BlastX (e-value cutoff: e-10) against the genes for use in studying pathways involved in resistance Cryphonectria parasitica proteome, significant matches to the chestnut blight. were found for 102 (~1.6%) and 213 (~1.5%) of the American and Chinese chestnut unigenes, respectively. Results 454 sequence from Canker tissue libraries 454 sequence from healthy stem tissue libraries The American Chestnut Canker cDNA library was con- Four libraries were constructed from American and Chi- structed from a pool of RNA isolated from canker tissues nese chestnut healthy stem tissues. For Chinese chestnut, of several individuals of one genotype (BA69). The Chi- two separate libraries were constructed from healthy cam- nese chestnut cDNA library was also prepared from RNA bial tissue collected from blight resistant genotypes 'Nan- extracted from several individuals of a single genotype king' and 'Mahogany'. For American chestnut two libraries (Nanking). One plate of sequencing was conducted with were constructed from the genotypes Watertown and Wis- each library using the GS20 model of the 454 system. A niewski. In contrast to the canker transcriptome sequenc- total of 129,508 and 235,635 reads were generated from ing, American chestnut and Chinese chestnut healthy American and Chinese chestnut canker transcriptomes stem transcriptomes were sequenced using the FLX system respectively, in the GS20 runs (NCBI SRA accessions (Roche). A quarter plate of sequencing was conducted for SRX001804 and SRX001799, respectively). The average each American chestnut healthy stem library, while a three length of the reads was 101 nucleotides (nt) (Table 1). The quarter plate worth of sequencing were conducted for the difference in the number of reads generated for the Amer- Chinese chestnut Nanking and Mahogany libraries (Table ican and the Chinese chestnut canker reflects the lower 1). Sequencing of the healthy stem transcriptome from quality of the American chestnut library. In total ~13.3 the two American chestnut genotypes yielded a total of and ~24.0 megabases of cDNA were generated from the 188,334 reads (NCBI SRA accessions SRX001800 and American and Chinese chestnut canker libraries, respec- SRX001801, respectively), with an average read length of tively. Prior to assembly, the canker raw sequence data ~246 nt (Table 1). Slightly more than 2.5 times that from the GS20 was re-analyzed with the improved base- number of reads (488,453) was generated from the two calling software of the new FLX model 454 sequencer. Chinese chestnut healthy stem genotypes (NCBI SRA Contig construction of the 454 reads using the Newbler accessions SRX001805 and SRX001806, respectively) Table 1: Summary of 454 sequencing results obtained in this study for the American and Chinese chestnut transcriptomes Sample System # Plates # of Reads # of bp AL of Reads # Contigs AL of All # Large AL of Large Contigs Contigs Contigs ACCanker GS20 1 129,508 13,080,308 101 7,171 168 247 689 ACHS1 FLX 3/4 126,791 29,828,910 247 11,496 276 885 817 ACHS2 FLX 3/4 162,624 38,165,054 246 9,431 271 691 816 CCCanker GS20 1 235,635 23,799,135 101 14,308 168 436 773 CCMHS FLX 3/4 228,594 56,051,191 246 21,828 344 3,074 851 CCNHS FLX 3/4 259,859 64,271,926 247 28,784 339 4,451 848 Total both 5 2,184,941 428,799,020 198 93.018 261 9,784 799 #, Number; AL, Average length ACCanker, American chestnut genotype BA69 infected stem (canker) cDNA CCCanker, Chinese chestnut variety 'Nanking' infected stem (canker) cDNA CCMHS, Chinese Chestnut variety 'Mahogany' healthy stem cDNA CCNHS, Chinese Chestnut variety 'Nanking' healthy stem cDNA ACHS1, American Chestnut Wisneiwski genotype healthy stem cDNA ACHS2, American Chestnut Watertown genotype healthy stem cDNA Page 3 of 11 (page number not for citation purposes) BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 with an average read length of 247.9 bases (Table 1). In Transcriptome comparison between canker and healthy total, ~46.4 and ~120 megabases of healthy stem tran- stem tissues within Chinese and American chestnut To determine the effect of the infection by the blight caus- scriptome sequence were obtained from American and Chinese chestnut healthy stems, respectively. We gener- ing fungus on gene expression in American and Chinese ated 20,927 contigs for American chestnut healthy stem chestnut trees, we compared the transcriptomes from can- and 50,612 contigs for Chinese chestnut healthy stem kered versus healthy stems (Fig. 2 and Fig. 3). We first using the Newbler assembly software package (454 Life determined how many times a unigene was represented in Sciences) with an average length of 273 nt and 330 nt, each of the libraries based on the number of reads for each respectively. A total of 1,823 and 7,961 contigs from (unigene count). We then determined which genes were American and Chinese chestnut, respectively, were con- in common in the two transcriptomes, versus being spe- sidered large with an average length of 833 nt. This left cific to a library, based on searching the GenBank Acces- 95,483 and 100,779 unassembled singleton reads from sion numbers of the contigs and reads as annotated by American and Chinese chestnut healthy stem sequences, BlastX alignment to the Arabidopsis proteome. Analysis of respectively. From the contigs, a total of 12,883 and canker and healthy stem transcriptomes from American 15,085 genes were tagged from American chestnut and and Chinese chestnut showed that several resistance- Chinese chestnut healthy stem tissues, respectively. related genes were differentially expressed in canker tis- sues (see Additional file 1 and Additional file 2). Those Functional annotation of American and Chinese chestnut genes encode various transcription factors such as WRKY, To determine the possible functions of genes tagged, we zinc finger, Myb, C2 domain, basic helix-loop-helix, used the Gene Ontology (GO) [25] classification system. CCAAT-box, and CCR4-NOT. Several other genes Based on the Arabidopsis proteome, a function could be involved in resistance to biotic stresses were differentially assigned to 83,292 (26%) and 20,391 (28%) of the 454 expressed in canker tissues. Those genes include cin- reads from American and Chinese chestnut respectively. namoyl-CoA reductase (CCR), 4-coumarate–CoA ligase, These percentages are lower than those obtained by hydrolase, kinases, phosphatases, translation factor, ATPases, BLASTx alignments to the Populus proteome (39% and pathogen responsive alpha-dioxygenase, etc (see Additional 46% for American and Chinese chestnut respectively). file 3 and Additional file 4). Some genes such as ABC However, most of the reads (45,804 and 139,230 for transporter, CCAAAT-box, CCR4-NOT and zinc finger seem American and Chinese chestnut respectively) with best to be differentially expressed in canker vs. healthy stem tis- hits to the Populus proteome are for Populus genes that are sues in Chinese chestnut. annotated as having no known function. GO ontology Transcriptome comparison between Chinese and analysis based on the Arabidopsis proteome showed that the distributions of gene functions for cDNA sequences American chestnut canker tissues from American and Chinese chestnut cankers are similar To gain insight into the differences in the response of the (Fig. 1). This expected result indicates that there is no bias American and Chinese chestnut species to infection by the in the construction of the libraries from American and blight-causing agent, we compared the transcriptomes Chinese canker tissues. The functions of genes identified from Chinese chestnut canker tissues and American chest- cover various biological processes. However, hydrolase nut canker tissues (Fig. 1) as described above. This com- and transferase are among the most represented molecu- parison showed that the distribution of gene functions lar function categories. The biological processes most rep- was very similar overall in both the cankers of both spe- resented were transport and protein metabolism. It is cies. However, we observed a small increase in nucleic noteworthy that a larger number of genes involved in acid protein binding and transcription factor molecular response to biotic and abiotic stimuli and stresses were functions in Chinese chestnut cankers. In an opposite pat- identified in Chinese chestnut tissues compared with tern, American chestnut had a small increase in the cate- American chestnut. This difference may be associated with gory "structural molecule activity". We also observed that blight resistance in Chinese chestnut. A similar pattern of the fraction of genes involved in response to stress and GO-annotation function distribution was found when the biotic and abiotic stimuli was slightly higher in American transcriptomes from healthy stem and canker tissues from chestnut canker tissue. However, statistical analysis using American and Chinese chestnut were compared (Fig. 2 GOstat program showed that none of those differences and Fig. 3). As predicted, we observed that the fraction of were statistically significant [26]. Detailed comparison of genes involved in response to stress, biotic and abiotic the transcriptomes showed that many resistance-related stimuli, cell organization and biogenesis processes are genes were differentially expressed in both the American highly represented. The molecular functions most repre- and the Chinese chestnut infection sites (see Additional sented are transferase, protein binding, and hydrolase. file 3 and Additional file 4). Statistical tests as per [27] of the expression data showed that the differential expres- Page 4 of 11 (page number not for citation purposes) BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 AC Canker CC Canker H ch Figure 1 iestnut ca stogram p nker resen tissues a tation of n Ge d biological ne Onto processe logy classification of putative molecular s in which they are involved function of unigenes from American and Chinese Histogram presentation of Gene Ontology classification of putative molecular function of unigenes from American and Chinese chestnut canker tissues and biological processes in which they are involved. sion of many of the resistance-related genes was statisti- used to analyze the transcriptome in non model plants. cally significant. Examples of the resistance-related genes However, deep EST sequencing using capillary sequenc- preferentially expressed in American chestnut (based on ing, which requires cDNA cloning and individual DNA the difference in reads per unigene) include genes encod- preparations for each clone, is time consuming and very ing proteins such as SNF7, laccase, CCR, cinnamyl alcohol costly. Bead-based pyrosequencing introduced recently dehydrogenase (CAD), expansin, F-box proteins, FAD- [18] constitutes a better alternative for transcriptomics. binding protein, proteins named disease-resistance- The high number of reads generated per run together with responsive, etc. Most of those genes play an important the low sequencing error rate in the contigs obtained role in plant response to pathogen infection. Genes pre- makes it a good tool to deeply sequence the transcriptome senting relatively high expression in Chinese chestnut of plants. This approach has been used successfully for encode proteins such as mitogen-activated kinase, Myb analyzing the transcriptomes of maize and Arabidopsis transcription factors, pathogen-responsive alpha-dioxyge- [19-22] and we have applied it to the non-model tree spe- nase, laccase, cytochrome P450, F-box proteins, SNF7, cies Castanea dentata and C. mollissima. CCR, succinyl-CoA ligase, etc. However, most of the gene expression differences in Chinese chestnut were not statis- Before this project, only a few hundred chestnut tically significant in our data set. sequences had been deposited in the EST database (dbEST) at NCBI. The data presented here represent the first large effort by the Fagaceae Genomic Tools Develop- Discussion American and Chinese chestnut transcriptome sequencing ment project to generate cDNA resources and analyze the Advances in DNA sequencing technology during the last transcriptomes of American and Chinese chestnut. These decade have dramatically impacted genome sequencing resources are public and the sequences can be accessed in and transcriptome analysis. Techniques such as microar- a searchable database at the project website [17], or as raw rays and SAGE have facilitated transcriptome analysis at sequence data at the NCBI Short Read Archive (accession large scale from numerous plants. However, those tech- above). In total, our study generated 171 Mb and 78 Mb niques could be used only for model plants with known and tagged 40,039 and 28,890 genes from Chinese chest- genome sequences. EST sequencing has been successfully nut and American chestnut, respectively. A fraction (rang- Page 5 of 11 (page number not for citation purposes) cellular component unknown other membranes other cellular components other intracellular components chloroplast mitochondria other cytoplasmic components nucleus ribosome plastid cytosol plasma membrane cell wall golgi apparatus ER extracellular DNA or RNA binding hydrolase activity kinase activity nucleic acid binding nucleotide binding other binding other enzyme activity other molecular function protein binding structural molecule activity transcription factor activity transferase activity transporter activity unknown molecular function receptor binding or activity cell organization and biogenesis developmental processes DNA or RNA metabolism electron transport or energy pathways other biological processes other cellular processes other metabolic processes protein metabolism response to abiotic or biotic stimulus response to stress signal transduction transcription transport unknown biological processes BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 AC Canker AC Healthy stem H h Figure 2 eistogram presentation of Gen althy stem and canker tissues and biolog e Ontology classifica ical procetion of putative molecular functions sses in which they are involved of unigenes from American chestnut Histogram presentation of Gene Ontology classification of putative molecular functions of unigenes from American chestnut healthy stem and canker tissues and biological processes in which they are involved. ing between 14% and 21%) of American and Chinese also include a large number of genes known to be chestnut unigenes that could not be annotated using the involved in response to biotic and abiotic stimuli and Arabidopsis proteome could however be annotated using stress in general. These gene sequences constitute a very the Populus proteome. Most of the genes with no hits to important resource to the scientific community working the Arabidopsis proteome encoded proteins annotated on chestnut blight resistance as well as those interested in with unknown functions in the poplar genome, however. gene discovery in Fagaceae species. Those genes could correspond to either tree specific genes or sequences that have diverged in Populus and chestnut By taking into consideration only the sequences that have beyond recognizable homology to Arabidopsis using the homologies in the Arabidopsis proteome, two plates of 454 Blast algorithm. Moreover, over 50% of the 454 reads sequences from American chestnut and Chinese chestnut could not be annotated using either the Arabidopsis pro- were enough to generate ~13,000 and ~15,000 unigenes teome or the Populus proteome. A query against the Fungi from each species. This number represents 52% and 60% database at NCBI excluded a Cryphonectria origin for a of American and Chinese chestnut transcriptome respec- small fraction of those reads (~3% for both species). tively, assuming that the two chestnut species have a sim- While a fraction of the remaining sequences may corre- ilar gene number as Arabidopsis. Such breadth and depth spond to 3' or 5' untranslated regions, non coding RNAs, (the number of reads per gene varying between 60 and or short sequences not containing a known protein 178) of coverage by 454 gene tagging, makes this tech- domain, a large number may correspond to potential nique a good tool for quantifying the expression level of Chestnut-specific genes. A similar situation was found sets of genes involved in various developmental stages or when analyzing the transcription of Eschscholzia califor- physiological conditions. cDNA sequences generated nica, Persea americana, and Aristolochia fimbriata [28] and from both species cover various biological processes and (Kerr Wall, personal communication). The two sets of molecular functions indicating that 454 sequencing con- unigenes from Chinese chestnut and American chestnut stitutes a powerful tool for sequencing the transcriptome Page 6 of 11 (page number not for citation purposes) cellular component unknown other membranes other cellular components other intracellular components chloroplast mitochondria other cytoplasmic components nucleus ribosome plastid cytosol plasma membrane cell wall golgi apparatus ER extracellular DNA or RNA binding hydrolase activity kinase activity nucleic acid binding nucleotide binding other binding other enzyme activity other molecular function protein binding structural molecule activity transcription factor activity transferase activity transporter activity unknown molecular function receptor binding or activity cell organization and biogenesis developmental processes DNA or RNA metabolism electron transport or energy pathways other biological processes other cellular processes other metabolic processes protein metabolism response to abiotic or biotic stimulus response to stress signal transduction transcription transport unknown biological processes BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 CC Canker CC Healthy stem h Figure 3 Heistogram presentation of Gen althy stem and canker tissues and biolog e Ontology classificati ical processe on of putative molecula s in which they are invo r functions lved of unigenes from Chinese chestnut Histogram presentation of Gene Ontology classification of putative molecular functions of unigenes from Chi- nese chestnut healthy stem and canker tissues and biological processes in which they are involved. of non model species. These results confirm that pyrose- members (unigenes or reads). Two members of the hydro- quencing constitutes a powerful tool for transcriptome lase group are the glycosyl hydrolase family 3 proteins, characterization and gene discovery. each of which was found five times in our transcriptome data. Glycosyl hydrolases break the bonds between carbo- Transcriptome comparison between canker tissues from hydrates and are involved in expansion and degradation Castanea mollissima and Castanea dentata of cell walls [30]. It is possible that some of the genes with GO annotation analyses showed that, overall, canker tis- hydrolase activity identified in Chinese chestnut canker sues from both species present a similar transcriptome. tissue are contaminants from the pathogen fungi (Cryph- Gene function categories associated with metabolic proc- onectria) mycelium within the canker and function by ess are highly represented in both transcriptomes. The cat- weakening the plant cell wall to facilitate fungal entry. egory represented the most is composed of genes However, analysis of these hydrolase sequences showed associated with various metabolic processes as previously that they are more similar to other plant hydrolase genes described in other systems such as cassava [29]. The sec- than to fungal hydrolase sequences. This suggests that ond most highly represented category includes genes these hydrolase proteins are of plant origin and function involved in resistance to stress and response to biotic and either by strengthening cell walls against pathogen entry abiotic stimuli. Detailed analysis of the 454 sequences or in the programmed cell death response of the cells at from both Chinese and American chestnut showed that the fungal infection site in the chestnut stem. A second the tagged genes included a large number associated with functional category well represented is kinase activity. resistance to biotic and abiotic stresses. These include Such genes are involved in signaling in pathogen infection genes involved in pathogen recognition and signaling, and play a key role in plant defense response. A third func- transcription factors, and resistance genes. Comparison of tional category observed in the chestnut transcriptomes is genes highly expressed in the canker tissues of both Amer- represented by transcription factors or genes associated ican and Chinese chestnut showed that a fraction were with RNA or protein binding. Such genes may modulate either preferentially expressed in American chestnut or in the expression of resistance genes in response to the path- Chinese chestnut. Genes with hydrolase activity repre- ogen infection. sented the functional category with the largest number of Page 7 of 11 (page number not for citation purposes) Relative number of genes, % cellular component unknown other membranes other cellular components other intracellular components chloroplast mitochondria other cytoplasmic components nucleus ribosome plastid cytosol plasma membrane cell wall golgi apparatus ER extracellular DNA or RNA binding hydrolase activity kinase activity nucleic acid binding nucleotide binding other binding other enzyme activity other molecular function protein binding structural molecule activity transcription factor activity transferase activity transporter activity unknown molecular function receptor binding or activity cell organization and biogenesis developmental processes DNA or RNA metabolism electron transport or energy pathways other biological processes other cellular processes other metabolic processes protein metabolism response to abiotic or biotic stimulus response to stress signal transduction transcription transport unknown biological processes BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 Candidate genes involved in chestnut response to the gene PAL2, a key enzyme in phenylpropanoid and Cryphonectria parasitica infection lignin biosynthesis [51]. WRKY transcription factors have Among genes that were found to be differentially been shown to fine tune the response of plants to chal- expressed in American or Chinese chestnut or both, sev- lenge with pathogens [52]. SNF genes interact with other eral are known to be involved in various processes of plant genes, such as SnRK1, which regulate glucose metabolism, defense against pathogens such as cell death related to cell defense and other cellular processes. hypersensitivity response, construction of a physical bar- rier to block the pathogen progression, as well as systemic Overall, this study allowed us to conclude that chestnut resistance. Among genes involved in hypersensitivity cell trees respond to Cryphonectria parasitica infection by acti- death, we found ABC transporter, C2-domain-containing vating both local and systemic resistance responses. The gene, methylenetetrahydrofolate reductase, elongation factor-1 trees try first to block the progression of the pathogen by alpha, and peroxidase. Such genes are involved in control- increasing the expression of hydrolases, lignin synthesis, ling the extent of the cell death in the defense response and cell death. The infection is also sensed by mitogen [31-34]. Pleiotropic drug resistance genes (ABC trans- kinases, which activate other transcriptions factors such as porter family), which are involved in jasmonic acid path- AP2, Myb, and WRKY, which in turn induce the expression way response, induce the secretion of secondary of genes from the phenylpropanoid, jasmonic acid, oligo- metabolites such as diterpenes that inhibit the growth of chitosan, and other pathways that are involved in resist- invading organisms [35-37]. The other category of genes ance to pathogens [53,54]. that seems to be involved in plant resistance to the patho- gen encodes proteins involved in lignin biosynthesis such Conclusion as CCR, CAD, o-methyltransferase 1, cytochrome P450, 4- In conclusion, this study allowed us to (i) Obtain over coumarate–CoA ligase, succinyl-CoA ligase, S-adenosyl- 28,000 and 40,000 unigenes from American and Chinese methionine synthase 3, and S-adenosylmethionine syn- chestnut, (ii) Compare the transcriptomes of American thase 2. Previous studies [38-42] showed that genes and Chinese chestnut following infection by Cryphonectria involved in lignin synthesis are over-expressed in various parasitica, (iii) Identify potential pathways involved in plants when they were challenged with pathogens. chestnut resistance to the Cryphonectria parasitica, and (iv) Among other resistance genes over-expressed in American Identify several candidate genes for resistance to necro- and Chinese chestnut, we found several laccase genes, trophic fungal pathogens in trees. which also belong to the phenylpropanoid pathway. Polyphenol oxidases (PPO) catalyzing the oxygen- Methods American and Chinese chestnut materials dependent oxidation of phenols to quinines, have been demonstrated to increase tomato plant resistance against Healthy cambial tissue was collected from the American Pseudomonas syringae [43]. We also found several ATP- chestnut genotypes 'Watertown' and "Wisniewski' grow- binding cassette transport proteins, which are involved in ing at the Connecticut Agricultural Experiment Station, both constitutive and jasmonic acid-dependent induced Lockwood Farm, Hamden CT. Canker tissue was collected defense [35]. Chestnut plants seem also to activate the from the American chestnut genotype BA69 growing at expression of genes involved in systemic resistance when The American Chestnut Foundation Meadowview they are challenged with the blight fungus. Among genes Research Farm, Meadowview VA. Healthy cambial tissue belonging to this pathway, we identified omega-3 fatty acid was collected from the Chinese chestnut blight resistant desaturase, suppressor of fatty acid desaturase deficiency (SFD1 genotypes 'Nanking' and 'Mahogany' growing at The and SFD2), Ras-related GTP-binding which are required for American Chestnut Foundation Meadowview Research systemic resistance [44,45]. ATPase was found to be over- Farm, Meadowview VA. Canker tissues were collected expressed in American and Chinese chestnut. This gene is from Chinese chestnut genotype 'Nanking' growing at required for the attenuation of the hypersensitive Meadowview Research Farm. To create cankers, the stems response [43]. Among genes involved in signaling, we of Chestnut trees were inoculated with the hypervirulent found several genes such as mitogen activated protein. This Cryphonectria strain EP155 as described by Hebard and protein kinase activates both local resistance and basal collaborators [55]. Canker tissues were sampled 5 and 14 resistance [38,46,47]. It also appears from our data that days post-inoculation and pooled before RNA prepara- metabolic flux may be involved in the chestnut resistance tion. All samples were collected in liquid nitrogen and fro- to the fungus. Several other genes involved in the regula- zen at -80°C until use. tion of resistance gene expression such as Acetyl co-enzyme A carboxyltransferase (CAC3), SNF, and several transcrip- RNA preparation and cDNA library synthesis tion factors such as WRKY, Zinc finger, Myb, etc were iden- Total RNA was prepared by the method of Chang and col- tified. Myb genes are involved in regulation of disease laborators [56]. Three to five grams of frozen tissue were resistance genes [48-50]; they regulate the expression of weighed, ground to a fine powder under liquid nitrogen, Page 8 of 11 (page number not for citation purposes) BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 and dispersed in CTAB buffer. Following 2 chloroform nut canker and Chinese chestnut canker tissues as well as extractions, RNA was precipitated with LiCl , again between canker and healthy stem tissues within each spe- extracted with chloroform and precipitated with ethanol. cies was done using test developed by Dr. Claverie's team The resulting RNA pellet was resuspended in 40–100 μl of [27]. DEPC-treated water, and the quality was assessed with an Agilent Technologies 2100 Bioanalyzer (Agilent Technol- Abbreviations ogies). Poly(A) RNA was then separated from total RNA CAD: cinnamyl alcohol dehydrogenase; CCR: cinnamyl- using the Poly(A) Purist kit (Ambion) and the quality CoA reductase; cDNA: complementary DNA; EST: assessed with an Agilent Technologies 2100 Bioanalyzer expressed sequence tag; GO: Gene Ontology; Mb: mega- (Agilent Technologies). cDNA was synthesized from the bases; NCBI: National Center for Biotechnology Informa- mRNA using the Just cDNA kit (Stratagene) using random tion; nt: nucleotide; SAGE: Serial Analysis of Gene hexamer primers provided with the kit to obtain better 5' Expression. to 3' coverage of transcripts than is possible using Poly(A) priming alone. Authors' contributions AB contributed to extracting RNA and making the 454 The resulting cDNA was used to construct a 454 library libraries, curated and analyzed the data, supervised the following the supplier's instructions (Roche Diagnostics). work SC and YZ, and wrote the paper. CS and YZ contrib- The sequencing was conducted at Penn State University uted to the bioinformatics analyses. DSD collected tissue using an FLX model 454 DNA sequencer (Roche Diagnos- samples, prepared RNA, cDNA and 454 libraries, and tics). helped prepare the first draft of the manuscript. KB and WAP contributed to the RNA preparation and discussion 454 library construction and sequencing of disease response gene candidates. NW manages all 454 libraries were constructed as described previously aspects of the Genomic Tool Development for the [57]. In summary, cDNAs were sheared by nebulization to Fagaceae Project. RS is the Principle Investigator of the yield fragments approximately 500 bp in length. Adaptor Genomic Tool Development for the Fagaceae Project and sequences were ligated to fragmented cDNA, which were was responsible for oversight, budget, obtaining the fund- subsequently immobilized on beads. The DNA fragments ing for the project, and contributing advice at each step of were then denatured to yield a single stranded DNA the research. This work was conducted in the laboratory of library which was amplified by emulsion PCR for JC, who initiated the research with American Chestnut sequencing. Sequencing of the library was performed on a Foundation funding, co-directs the 454 sequencing facil- GS20 and an FLX model 454 DNA sequencer (454 Life ity at Penn State, and contributed to the development of Sciences). All raw 454 sequence data generated in this 454 sequencing protocols, evaluation and discussion of study is available at the Short Read Archive at the National the results, and preparation of the manuscript. Center for Biotechnology Information [24], specifically NCBI accession numbers SRX001799, SRX001800, Additional material SRX001801, SRX001804, RX001805, and SRX001806 (submission SRP000395). Additional File 1 Genes more highly expressed in canker tissues than healthy stem tis- Transcript Assembly and analysis sues of American Chestnut. The data from the 454 read sequences were assembled Click here for file into transcript contigs using Newbler Assembler software [http://www.biomedcentral.com/content/supplementary/1471- 2229-9-51-S1.docx] (Roche). Reads from each library were assembled sepa- rately. The unigene (contigs and remaining unique single- Additional File 2 tons) sequences were annotated by query against the Genes more highly expressed in canker tissues than in healthy stem tis- proteomes of Arabidopsis [58]) and Populus [59] and the sues of Chinese Chestnut. predicted proteome for the blight fungus Cryphonectria Click here for file parasitica [60] using Blastx (e-value cutoff of -10). The [http://www.biomedcentral.com/content/supplementary/1471- Gene Ontology (GO) (Consortium, 2008) system was 2229-9-51-S2.docx] used to summarize possible functional classifications of Additional File 3 the unigenes via assignment of Arabidopsis gene identifiers Disease and Defense Response Genes More Highly Expressed in with the strongest BLASTx alignments to the correspond- Infected Tissues of Chinese Chestnut (CC) than in American Chestnut ing chestnut 454 reads. Comparison of the distribution of (AC). biological processes or molecular function obtained using Click here for file Go annotation was done using GOstat program [26]. [http://www.biomedcentral.com/content/supplementary/1471- Comparison of gene expression between American chest- 2229-9-51-S3.doc] Page 9 of 11 (page number not for citation purposes) BMC Plant Biology 2009, 9:51 http://www.biomedcentral.com/1471-2229/9/51 15. Carraway DT, Merkle SA: Plantlet regeneration fomr somatic embryos of American Chestnut. Can J For Res 1997, Additional File 4 27:1805-1812. Disease- and Defense- Response Genes More Highly Expressed in 16. Xing Z, Powell WA, Maynard CA: Development and germina- Infected Tissues of American Chestnut (AC) than in Chinese Chestnut tion of American chestnut somatic embryos. Plant Cell, Tissue and Organ Culture 1999, 57:47-55. (CC). 17. The Fagaceae Genomic Tools Project [http:// Click here for file www.fagaceae.org] [http://www.biomedcentral.com/content/supplementary/1471- 18. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, 2229-9-51-S4.doc] Berka J, Braverman MS, Chen YJ, Chen Z, et al.: Genome sequenc- ing in microfabricated high-density picolitre reactors. Nature 2005, 437(7057):376-380. 19. Weber AP, Weber KL, Carr K, Wilkerson C, Ohlrogge JB: Sampling the Arabidopsis transcriptome with massively parallel pyro- Acknowledgements sequencing. Plant Physiol 2007, 144(1):32-42. 20. Redestig H, Weicht D, Selbig J, Hannah MA: Transcription factor We would like to thank Dr. Sandra L. Anagnostakis and Dr. Fredrick V. target prediction using multiple short expression time series Hebard for providing us with chestnut tissues. We also thank Dr. Haiying from Arabidopsis thaliana. BMC Bioinformatics 2007, 8:454. Liang for her help with cDNA library preparation as well as our colleagues 21. Ohtsu K, Smith MB, Emrich SJ, Borsuk LA, Zhou R, Chen T, Zhang X, Dr Stephan Schuster, Lynn Tomsho, and Michael Packard for 454 library Timmermans MC, Beck J, Buckner B, et al.: Global gene expression preparation and for expert technical assistance with 454 sequencing. We analysis of the shoot apical meristem of maize (Zea mays L.). Plant J 2007, 52(3):391-404. thank Kerr Wall, Alex Choi and Urmila Plakkat for their help with sequence 22. Emrich SJ, Barbazuk WB, Li L, Schnable PS: Gene discovery and analysis, tables and figure preparation. We thank the Joint Genome Institute annotation using LCM-454 transcriptome sequencing. for providing us with the Cryphonectria parasitica proteome and EST Genome Res 2007, 17(1):69-73. sequences. Our thanks also go to all the Genomic Tool Development for 23. Morozova O, Marra MA: Applications of next-generation sequencing technologies in functional genomics. Genomics the Fagaceae Project partners. This work was supported by grant DBI- 2008, 92(5):255-264. PGPR-TRPGR 0605135 from The National Science Foundation's Plant 24. National Center for Biotechnology Information [http:// Genome Research Program, The Schatz Center for Tree Molecular Genet- www.ncbi.nlm.nih.gov] ics, and The American Chestnut Foundation. 25. Consortium GO: The Gene Ontology project in 2008. Nucleic Acids Res 2008, 36:D440-444. 26. Beissbarth T, Speed TP: GOstat: find statistically overrepre- References sented Gene Ontologies within a group of genes. Bioinformatics 1. 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