The Dictyostelium developmental cDNA project: generation and analysis of expressed sequence tags from the first-finger stage of development.

In an effort to identify and characterize genes expressed during multicellular development ill Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.     

Discovery of Phytophthora infestans Genes Expressed in Planta through Mining of cDNA Libraries

Background

Phytophthora infestans (Mont.) de Bary causes late blight of potato and tomato, and has a broad host range within the Solanaceae family. Most studies of the Phytophthora – Solanum pathosystem have focused on gene expression in the host and have not analyzed pathogen gene expression in planta.

Methodology/Principal Findings

We describe in detail an in silico approach to mine ESTs from inoculated host plants deposited in a database in order to identify particular pathogen sequences associated with disease. We identified candidate effector genes through mining of 22,795 ESTs corresponding to P. infestans cDNA libraries in compatible and incompatible interactions with hosts from the Solanaceae family.

Conclusions/Significance

We annotated genes of P. infestans expressed in planta associated with late blight using different approaches and assigned putative functions to 373 out of the 501 sequences found in the P. infestans genome draft, including putative secreted proteins, domains associated with pathogenicity and poorly characterized proteins ideal for further experimental studies. Our study provides a methodology for analyzing cDNA libraries and provides an understanding of the plant – oomycete pathosystems that is independent of the host, condition, or type of sample by identifying genes of the pathogen expressed in planta.     

Variability of gene expression profiles in human blood and lymphoblastoid cell lines

Background

Infection of plants by pathogens and the subsequent disease development involves substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. We have employed expressed sequence tag (EST) analysis to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe oryzae and fungal genes involved in infectious growth within the host during a compatible interaction.

Results

A cDNA library was constructed with RNA from rice leaves (Oryza sativa cv. Hwacheong) infected with M. oryzae strain KJ201. To enrich for fungal genes, subtraction library using PCR-based suppression subtractive hybridization was constructed with RNA from infected rice leaves as a tester and that from uninfected rice leaves as the driver. A total of 4,148 clones from two libraries were sequenced to generate 2,302 non-redundant ESTs. Of these, 712 and 1,562 ESTs could be identified to encode fungal and rice genes, respectively. To predict gene function, Gene Ontology (GO) analysis was applied, with 31% and 32% of rice and fungal ESTs being assigned to GO terms, respectively. One hundred uniESTs were found to be specific to fungal infection EST. More than 80 full-length fungal cDNA sequences were used to validate ab initio annotated gene model of M. oryzae genome sequence.

Conclusion

This study shows the power of ESTs to refine genome annotation and functional characterization. Results of this work have advanced our understanding of the molecular mechanisms underpinning fungal-plant interactions and formed the basis for new hypothesis.     

A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries.

For comprehensive analysis of genes expressed in the model dicotyledonous plant, Arabidopsis thaliana, expressed sequence tags (ESTs) were accumulated. Normalized and size-selected cDNA libraries were constructed from aboveground organs, flower buds, roots, green siliques and liquid-cultured seedlings, respectively, and a total of 14,026 5'-end ESTs and 39,207 3'-end ESTs were obtained. The 3'-end ESTs could be clustered into 12,028 non-redundant groups. Similarity search of the non-redundant ESTs against the public non-redundant protein database indicated that 4816 groups show similarity to genes of known function, 1864 to hypothetical genes, and the remaining 5348 are novel sequences. Gene coverage by the non-redundant ESTs was analyzed using the annotated genomic sequences of approximately 10 Mb on chromosomes 3 and 5. A total of 923 regions were hit by at least one EST, among which only 499 regions were hit by the ESTs deposited in the public database. The result indicates that the EST source generated in this project complements the EST data in the public database and facilitates new gene discovery.     

Expressed sequence tags from the flower pathogen Claviceps purpurea

The ascomycete Claviceps purpurea (ergot) is a biotrophic flower pathogen of rye and other grasses. The deleterious toxic effects of infected rye seeds on humans and grazing animals have been known since the Middle Ages. To gain further insight into the molecular basis of this disease, we generated about 10 000 expressed sequence tags (ESTs)—about 25% originating from axenic fungal culture and about 75% from tissues collected 6–20 days after infection of rye spikes. The pattern of axenic vs. in planta gene expression was compared. About 200 putative plant genes were identified within the in planta library. A high percentage of these were predicted to function in plant defence against the ergot fungus and other pathogens, for example pathogenesis-related proteins. Potential fungal pathogenicity and virulence genes were found via comparison with the pathogen–host interaction database (PHI-base; http://www.phi-base.org ) and with genes known to be highly expressed in the haustoria of the bean rust fungus. Comparative analysis of Claviceps and two other fungal flower pathogens (necrotrophic Fusarium graminearum and biotrophic Ustilago maydis ) highlighted similarities and differences in their lifestyles, for example all three fungi have signalling components and cell wall-degrading enzymes in their arsenal. In summary, the analysis of axenic and in planta ESTs yielded a collection of candidate genes to be evaluated for functional roles in this plant–microbe interaction.     

The use of direct cDNA selection to rapidly and effectively identify genes in the fungus Aspergillus fumigatus

Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. To rapidly identify genes in this fungus, including potential targets for chemotherapy, diagnostics, and vaccine development, we constructed cDNA libraries. We began with non-normalized libraries, then to improve this approach we constructed a normalized cDNA library using direct cDNA selection. Normalization resulted in a reduction of the frequency of clones with highly expressed genes and an enrichment of underrepresented cDNAs. Expressed sequence tags generated from both the original and the normalized libraries were compared with the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, indicating that a large proportion of A. fumigatus genes do not have orthologs in these fungal species. This method allowed the expeditious identification of genes in a fungal pathogen. The same approach can be applied to other human or plant pathogens to rapidly identify genes without the need for genomic sequence information.     

Expressed sequence tag analysis of the soybean rust pathogen Phakopsora pachyrhizi

Soybean rust is caused by the obligate fungal pathogen Phakopsora pachyrhizi Sydow. A unidirectional cDNA library was constructed using mRNA isolated from germinating P. pachyrhizi urediniospores to identify genes expressed at this physiological stage. Single pass sequence analysis of 908 clones revealed 488 unique expressed sequence tags (ESTs, unigenes) of which 107 appeared as multiple copies. BLASTX analysis identified 189 unigenes with significant similarities (Evalue<10(-5)) to sequences deposited in the NCBI non-redundant protein database. A search against the NCBI dbEST using the BLASTN algorithm revealed 32 ESTs with high or moderate similarities to plant and fungal sequences. Using the Expressed Gene Anatomy Classification, 31.7% of these ESTs were involved in primary metabolism, 14.3% in gene/protein expression, 7.4% in cell structure and growth, 6.9% in cell division, 4.8% in cell signaling/cell communication, and 4.8% in cell/organism defense. Approximately 29.6% of the identities were to hypothetical proteins and proteins with unknown function.     

Expressed sequence tags from persimmon at different developmental stages

Persimmon (Diospyros kaki Thunb.) is an important fruit in Asian countries, where it is eaten as a fresh fruit and is also used for many other purposes. To understand the molecular mechanism of fruit development and ripening in persimmon, we generated a total of 9,952 expressed sequence tags (ESTs) from randomly selected clones of two different cDNA libraries. One cDNA library was derived from fruit of “Saijo” persimmon at an early stage of development, and the other from ripening fruit. These ESTs were clustered into 6,700 non-redundant sequences. Of the 6,700 non-redundant sequences evaluated, the deduced amino acid sequences of 4,356 (65%) showed significant homology to known proteins, and 2,344 (35%) showed no significant similarity to any known proteins in Arabidopsis databases. We report comparison of genes identified in the two cDNA libraries and describe some putative genes involved in proanthocyanidin and carotenoid synthesis. This study provides the first global overview of a set of genes that are expressed during fruit development and ripening in persimmon.     

Analysis of expressed sequence tags from Chaetomium cupreum grown under conditions associated with mycoparasitism

Aims:  To elucidate the molecular mechanisms associated with mycoparasitism from Chaetomium cupreum , an effective biocontrol agent with ability against plant pathogenic fungi.
Methods and Results:  One cDNA library was constructed from conditions predicted to resemble mycoparasitic process. A total of 1876 ESTs were generated and assembled into 1035 unigenes. B last X search revealed that 585 unigenes had similarities with sequences available from public databases. Based on the ESTs abundance, MFS monosaccharide transporter was found as the gene expressed at the highest level. A KEGG analysis allowed mapping of 60 metabolic pathways well represented by the glycolysis/gluconeogenesis, d -arginine and ornithine metabolism, and tryptophan metabolism. The genes related to mycoparasitism were detected.
Conclusions:  The results revealed that the cell walls of the fungal pathogen can simulate some aspects of the mycoparasitic interaction between C. cupreum and its targets.
Significance and Impact of the Study:  This is the first report to study genes expression under conditions associated with the mycoparasitic process. The findings contribute to elucidate the molecular mechanisms involved in mycoparasitism and will help to advance our efforts in developing novel strategies for biocontrol of plant fungal diseases.     

Analysis of expressed sequence tags from Gibberella zeae (anamorph Fusarium graminearum)

Gibberella zeae is a broad host range pathogen that infects many crop plants, including wheat and barley, and causes head blight and rot diseases throughout the world. To better understand fungal development and pathogenicity, we have generated 7996 ESTs from three cDNA libraries. Two libraries were generated from carbon-(C-) and nitrogen- (N-) starved mycelia and one library was generated from cultures of maturing perithecia (P). In other fungal pathogens, starvation conditions have been shown to act as cues to induce infection-related gene expression. To assign putative function to cDNAs, sequences were initially assembled using StackPack. The estimated total number of genes identified from the three EST databases was 2110: 1088 contigs and 1022 singleton sequences. These 2110 sequences were compared to a yeast protein sequence reference set and to the GenBank nonredundant database using BLASTX. Based on presumptive gene function identified by this process, we found that the two starved cultures had similar, but not identical, patterns of gene expression, whereas the developmental cultures were distinct in their pattern of expression. Of the three libraries, the perithecium library had the greatest percentage (46%) of ESTS falling into the "unclassified" category. Homologues of some known fungal virulence or pathogenicity factors were found primarily in the N- and C-libraries. Comparisons also were made with ESTs from the related fungi, Neurospora crassa and Magnaporthe grisea and the genomic sequence of N. crassa.     

Identification and characterization of differential gene expression in the mesocarp and kernel of oil palm nuts using suppression subtractive hybridization

Suppression subtracted hybridization (SSH) and dot blotting were used to identify differential gene expression in the mesocarp and kernel of oil palm nuts. The different types of nut tissue show differences in fatty acid anabolism and the synthesis of other important compounds. In total, 302 clones from forward SSH libraries and 238 clones from reverse SSH libraries were identified following differential screening, respectively. Among these, 120 clones from the forward SSH library and 81 clones from the reverse SSH library, showed tenfold or more differential expression levels, and were sequenced. Sequence analysis revealed that 76 clones (28 from the forward SSH library and 48 from the reverse SSH library) represent non-redundant cDNA inserts. The differential expression of 39 subset genes in the two different tissues was further confirmed by RT-PCR analysis. Functionally annotated blasting against the GenBank non-redundant protein database classified all 76 candidate genes into six categories, according to their putative functions. Interestingly, our results show that a group of significantly differentially expressed genes are involved in processes associated with oil palm nut maturation, such as the synthesis of medium-chain saturated fatty acids and phytic acid, nut development, and stress/defense responses. This study describes some relationships between gene expression and metabolic pathways in mature oil palm nuts, and contributes to our understanding of oil palm nut ESTs.     

Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger

In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×10(6) pfu/ml and 1.62×10(9) pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142 bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers.     

Gene expression profiling of the plant pathogenic basidiomycetous fungus Rhizoctonia solani AG 4 reveals putative virulence factors

Rhizoctonia solani is a ubiquitous basidiomycetous soilborne fungal pathogen causing damping-off of seedlings, aerial blights and postharvest diseases. To gain insight into the molecular mechanisms of pathogenesis a global approach based on analysis of expressed sequence tags (ESTs) was undertaken. To get broad gene-expression coverage, two normalized EST libraries were developed from mycelia grown under high nitrogen-induced virulent and low nitrogen/methylglucose-induced hypovirulent conditions. A pilot-scale assessment of gene diversity was made from the sequence analyses of the two libraries. A total of 2280 cDNA clones was sequenced that corresponded to 220 unique sequence sets or clusters (contigs) and 805 singlets, making up a total of 1025 unique genes identified from the two virulence-differentiated cDNA libraries. From the total sequences, 295 genes (38.7%) exhibited strong similarities with genes in public databases and were categorized into 11 functional groups. Approximately 61.3% of the R. solani ESTs have no apparent homologs in publicly available fungal genome databases and are considered unique genes. We have identified several cDNAs with potential roles in fungal pathogenicity, virulence, signal transduction, vegetative incompatibility and mating, drug resistance, lignin degradation, bioremediation and morphological differentiation. A codon-usage table has been formulated based on 14694 R. solani EST codons. Further analysis of ESTs might provide insights into virulence mechanisms of R. solani AG 4 as well as roles of these genes in development, saprophytic colonization and ecological adaptation of this important fungal plant pathogen.