Gene discovery and gene expression in the rice blast fungus, Magnaporthe grisea: analysis of expressed sequence tags

Over 28,000 expressed sequence tags (ESTs) were produced from cDNA libraries representing a variety of growth conditions and cell types. Several Magnaporthe grisea strains were used to produce the libraries, including a nonpathogenic strain bearing a mutation in the PMK1 mitogen-activated protein kinase. Approximately 23,000 of the ESTs could be clustered into 3,050 contigs, leaving 5,127 singleton sequences. The estimate of 8,177 unique sequences indicates that over half of the genes of the fungus are represented in the ESTs. Analysis of EST frequency reveals growth and cell type-specific patterns of gene expression. This analysis establishes criteria for identification of fungal genes involved in pathogenesis. A large fraction of the genes represented by ESTs have no known function or described homologs. Manual annotation of the most abundant cDNAs with no known homologs allowed us to identify a family of metallothionein proteins present in M. grisea, Neurospora crassa, and Fusarium graminearum. In addition, multiply represented ESTs permitted the identification of alternatively spliced mRNA species. Alternative splicing was rare, and in most cases, the alternate mRNA forms were unspliced, although alternative 5' splice sites were also observed.     

Expressed sequences from the basidiomycetous tree pathogen Heterobasidion annosum during early infection of scots pine

The pattern of gene expression of the basidiomycete Heterobasidion annosum, causal agent of the root rot of conifers, was analysed during its interaction with pine roots. A complementary DNA (cDNA) library was constructed from total RNA extracted from H. annosum mycelia challenged with Scots pine seedling roots for 6 and 72h. Single pass sequencing of 1148 randomly selected cDNA clones resulted in 923 expressed sequence tags (ESTs). Contig analysis and sequence comparisons identified 318 unigene sequences, of which 62 were repeatedly sampled. A putative cellular function was assigned to 223 contigs (70%) that showed a moderate to high homology to protein sequences from public databases. Variations in expression levels during the infection process were monitored on a set of 96 unigenes by reverse northern using dot hybridisation. Seven unigenes (7%) were shown to be either up (4) or down (3) regulated during interaction of the fungus with pine roots. Fungal genes differentially expressed during contact with roots include genes encoding mitochondrial proteins, a cytochrome P450 and a vacuolar ATP synthase.     

Sequence evaluation of four specific cDNA libraries for developmental genomics of sunflower

Four different cDNA libraries were constructed from sunflower protoplasts growing under embryogenic and non-embryogenic conditions: one standard library from each condition and two subtractive libraries in opposite sense. A total of 22,876 cDNA clones were obtained and 4800 ESTs were sequenced, giving rise to 2479 high quality ESTs representing an unigene set of 1502 sequences. This set was compared with ESTs represented in public databases using the programs BLASTN and BLASTX, and its members were classified according to putative function using the catalog in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Some 33% of sequences failed to align with existing plant ESTs and therefore represent putative novel genes. The libraries show a low level of redundancy and, on average, 50% of the present ESTs have not been previously reported for sunflower. Several potentially interesting genes were identified, based on their homology with genes involved in animal zygotic division or plant embryogenesis. We also identified two ESTs that show significantly different levels of expression under embryogenic and non-embryogenic conditions. The libraries described here represent an original and valuable resource for the discovery of yet unknown genes putatively involved in dicot embryogenesis and improving our knowledge of the mechanisms involved in polarity acquisition by plant embryos.Communicated by R. Hagemann     

Differential gene expression in abdomens of the malaria vector mosquito, Anopheles gambiae, after sugar feeding, blood feeding and Plasmodium berghei infection

Background

Large scale sequencing of cDNA libraries can provide profiles of genes expressed in an organism under defined biological and environmental circumstances. We have analyzed sequences of 4541 Expressed Sequence Tags (ESTs) from 3 different cDNA libraries created from abdomens from Plasmodium infection-susceptible adult female Anopheles gambiae. These libraries were made from sugar fed (S), rat blood fed (RB), and P. berghei-infected (IRB) mosquitoes at 30 hours after the blood meal, when most parasites would be transforming ookinetes or very early oocysts.

Results

The S, RB and IRB libraries contained 1727, 1145 and 1669 high quality ESTs, respectively, averaging 455 nucleotides (nt) in length. They assembled into 1975 consensus sequences - 567 contigs and 1408 singletons. Functional annotation was performed to annotate probable molecular functions of the gene products and the biological processes in which they function. Genes represented at high frequency in one or more of the libraries were subjected to digital Northern analysis and results on expression of 5 verified by qRT-PCR.

Conclusion

13% of the 1965 ESTs showing identity to the A. gambiae genome sequence represent novel genes. These, together with untranslated regions (UTR) present on many of the ESTs, will inform further genome annotation. We have identified 23 genes encoding products likely to be involved in regulating the cellular oxidative environment and 25 insect immunity genes. We also identified 25 genes as being up or down regulated following blood feeding and/or feeding with P. berghei infected blood relative to their expression levels in sugar fed females.     

The construction of Arabidopsis expressed sequence tag assemblies. A new resource to facilitate gene identification.

The generation of large numbers of partial cDNA sequences, or expressed sequence tags (ESTs), has provided a method with which to sample a large number of genes from an organism. More than 25,000 Arabidopsis thaliana ESTs have been deposited in public databases, producing the largest collection of ESTs for any plant species. We describe here the application of a method of reducing redundancy and increasing information content in this collection by grouping overlapping ESTs representing the same gene into a "contig" or assembly. The increased information content of these assemblies allows more putative identifications to be assigned based on the results of similarity searches with nucleotide and protein databases. The results of this analysis indicate that sequence information is available for approximately 12,600 nonoverlapping ESTs from Arabidopsis. Comparison of the assemblies with 953 Arabidopsis coding sequences indicates that up to 57% of all Arabidopsis genes are represented by an EST. Clustering analysis of these sequences suggests that between 300 and 700 gene families are represented by between 700 and 2000 sequences in the EST database. A database of the assembled sequences, their putative identifications, and cellular roles is available through the World Wide Web.     

Analysis of Expressed Sequence Tags in Porcine Uterus Tissue

Two non-normalized cDNA libraries of uteri from Danish Landrace and Chinese Erhualian pigs were constructed, and 13,756 expressed sequence tags (ESTs) were randomly sequenced. The ESTs were clustered by Phrap software, and 6,139 distinct tentative consensus sequences were produced, including 2,730 contigs and 3,409 singlets. Using Blast tools, these 6,139 candidate genes were compared to the nr and nt databases; 5,210 of them were assigned putative functions, whereas 929 potentially represent new genes. Highly expressed genes appear to be associated with basic energy metabolism, transferase activity, localization, cellular physiological process, protein binding, and nucleic acid binding. Antileukoproteinase was the most highly expressed gene, corresponding to endometrial differentiation and conceptus or fetal development.     

Gene expression in autumn leaves

Two cDNA libraries were prepared, one from leaves of a field-grown aspen (Populus tremula) tree, harvested just before any visible sign of leaf senescence in the autumn, and one from young but fully expanded leaves of greenhouse-grown aspen (Populus tremula x tremuloides). Expressed sequence tags (ESTs; 5,128 and 4,841, respectively) were obtained from the two libraries. A semiautomatic method of annotation and functional classification of the ESTs, according to a modified Munich Institute of Protein Sequences classification scheme, was developed, utilizing information from three different databases. The patterns of gene expression in the two libraries were strikingly different. In the autumn leaf library, ESTs encoding metallothionein, early light-inducible proteins, and cysteine proteases were most abundant. Clones encoding other proteases and proteins involved in respiration and breakdown of lipids and pigments, as well as stress-related genes, were also well represented. We identified homologs to many known senescence-associated genes, as well as seven different genes encoding cysteine proteases, two encoding aspartic proteases, five encoding metallothioneins, and 35 additional genes that were up-regulated in autumn leaves. We also indirectly estimated the rate of plastid protein synthesis in the autumn leaves to be less that 10% of that in young leaves.