Exploration of the late stages of the tomato-Phytophthora parasitica interactions through histological analysis and generation of expressed sequence tags.

The oomycete Phytophthora parasitica is a soilborne pathogen infecting numerous plants. The infection process includes an initial biotrophic stage, followed by a necrotrophic stage. The aim here was to identify genes that are involved in the late stages of infection. Using the host tomato and a transformed strain of P. parasitica expressing the green fluorescent protein (GFP), the various infection steps from recognition of the host to the colonization of plant tissues were studied. This late stage was selected to generate 4000 ESTs (expressed sequence tags), among which approx. 80% were from the pathogen. Comparison with an EST data set created previously from in vitro growth of P. parasitica allowed the identification of several genes, the expression of which might be regulated during late stages of infection. Changes in gene expression of several candidate genes predicted from in silico analysis were validated by quantitative RT-PCR experiments. These results give insights into the molecular bases of the necrotrophic stage of an oomycete pathogen.     

Single nucleotide polymorphisms in cytochrome P450 genes from barley

Plant cytochrome P450s are known to be essential in a number of economically important pathways of plant metabolism but there are also many P450s of unknown function accumulating in expressed sequence tag (EST) and genomic databases. To detect trait associations that could assist in the assignment of gene function and provide markers for breeders selecting for commercially important traits, detection of polymorphisms in identified P450 genes is desirable. Polymorphisms in EST sequences provide so-called perfect markers for the associated genes. The International Triticeae EST Cooperative data base of 24,344 ESTs was searched for sequences exhibiting homology to P450 genes representing the nine known clans of plant P450s. Seventy five P450 ESTs were identified of which 24 had best matches in Genbank to P450 genes of known function and 51 to P450s of unknown function. Sequence information from PCR products amplified from the genomic template DNA of 11 barley varieties was obtained using primers designed from six barley P450 ESTs and one durum wheat P450 EST. Single nucleotide polymorphisms (SNPs) between barley varieties were identified using five of the seven PCR products. A maximum of five SNPs and three haplotypes among the 11 barley lines were detected in products from any one primer pair. SNPs in three PCR products led to changes between barley varieties in at least one restriction site enabling genotyping and mapping without the expense of a specialist SNP detection system. The overall frequency of SNPs across the 11 barley varieties was 1 every 131 bases.     

Identification and characterization of differentially expressed ESTs of Gossypium barbadense infected by Verticillium dahliae with suppression subtractive hybridization

Cotton wilt defense reaction is a complicated continuous process and involves a battery of genes. In this study, we adopted suppression subtractive hybridization (SSH) technique to isolate differentially expressed ESTs from Gossypium barbadense variety 7124 during Verticillium wilt defense process. An array of 1165 clones from the subtractive library has been screened with reverse northern blotting, of which 131 ESTs were considered as over-expressed and 16 ESTs were down-regulated. Sequence analysis and blast search showed that 83 ESTs were homologous to 45 unique sequences in the databases. Among all these differentially expressed ESTs, at least three kinds of genes were characterized. The majority of ESTs with deduced identity to aerobic metabolism enzymes strongly expressed in the infection process. Likewise, ESTs similar to those reported for pathogen-related protein genes were also picked out in this study. These ESTs in combination with other kinase-like genes and a defensin-like EST constituted an assembly of genes responded during pathogens' infection. These results imply that sea-island cotton undergoes strong oxidative stress and results in a series of defense responses when attacked by V. dahliae. To our knowledge, this is the first report on the isolation of global ESTs during sea-island cotton defense reaction.     

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.     

A unigene catalogue of 5700 expressed genes in cassava

Two economically important characters, starch content and cassava bacterial blight resistance, were targeted to generate a large collection of cassava ESTs. Two libraries were constructed from cassava root tissues of varieties with high and low starch contents. Other libraries were constructed from plant tissues challenged by the pathogen Xanthomonas axonopodis pv.manihotis. We report here the single pass sequencing of 11 954 cDNA clones from the 5’ ends, including 111 from the 3’ ends. Cluster analysis permitted the identification of a unigene set of 5700 sequences. Sequence analyses permitted the assignment of a putative functional category for 37% of sequences whereas ~ 16% sequences did not show any significant similarity with other proteins present in the database and therefore can be considered as cassava specific genes. A group of genes belonging to a large multigene family was identified. We characterize a set of genes detected only in infected libraries putatively involved in the defense response to pathogen infection. By comparing two libraries obtained from cultivars contrasting in their starch content a group of genes associated to starch biosynthesis and differentially expressed was identified. This is the first large cassava EST resource developed today and publicly available thus making a significant contribution to genomic knowledge of cassava.     

Expressed sequence tags of Chinese cabbage flower bud cDNA.

We randomly selected and partially sequenced cDNA clones from a library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower bud cDNAs. Out of 1216 expressed sequence tags (ESTs), 904 cDNA clones were unique or nonredundant. Five hundred eighty-eight clones (48.4%) had sequence homology to functionally defined genes at the peptide level. Only 5 clones encoded known flower-specific proteins. Among the cDNAs with no similarity to known protein sequences (628), 184 clones had significant similarity to nucleotide sequences registered in the databases. Among these 184 clones, 142 exhibited similarities at the nucleotide level only with plant ESTs. Also, sequence similarities were evident between these 142 ESTs and their matching ESTs when compared using the deduced amino acid sequences. Therefore, it is possible that the anonymous ESTs encode plant-specific ubiquitous proteins. Our extensive EST analysis of genes expressed in floral organs not only contributes to the understanding of the dynamics of genome expression patterns in floral organs but also adds data to the repertoire of all genomic genes.     

Gene expression and EST analyses of Ustilago maydis germinating teliospores

Ustilago maydis grows in its host Zea mays eliciting the formation of obvious tumors that are full of black teliospores. Teliospores are thick-walled, dormant, diploid cells that have evolved for dispersal and survival of the pathogen. Their germination leads to new rounds of infection and is temporally linked to meiosis. We are investigating gene expression during teliospore germination to gain insight into the control of this process. Here we identify genes expressed through creation of an expressed sequence tag (EST) library. We generated 2871 ESTs that are assembled into 1293 contiguous sequences. Based upon a blast search similarity cutoff of E < or =10(-5) 38% of all contigs were orphans while 62% showed similarity to sequences in the protein database. Analyses of blast searches were used to functionally classify genes. Northern hybridizations using specific cDNA clones reveal a relative level of expression consistent with the number of sequences per contig. Identified genes and expression information provide a base for genome annotation of U. maydis and further investigation of teliospore germination and pathogenesis.     

Expressed sequence tags from phytophthora sojae reveal genes specific to development and infection

Six unique expressed sequence tag (EST) libraries were generated from four developmental stages of Phytophthora sojae P6497. RNA was extracted from mycelia, swimming zoospores, germinating cysts, and soybean (Glycine max (L.) Merr.) cv. Harosoy tissues heavily infected with P. sojae. Three libraries were created from mycelia growing on defined medium, complex medium, and nutrient-limited medium. The 26,943 high-quality sequences obtained clustered into 7,863 unigenes composed of 2,845 contigs and 5,018 singletons. The total number of P. sojae unigenes matching sequences in the genome assembly was 7,412 (94%). Of these unigenes, 7,088 (90%) matched gene models predicted from the P. sojae sequence assembly, but only 2,047 (26%) matched P. ramorum gene models. Analysis of EST frequency from different growth conditions and morphological stages revealed genes that were specific to or highly represented in particular growth conditions and life stages. Additionally, our results indicate that, during infection, the pathogen derives most of its carbon and energy via glycolysis of sugars in the plant. Sequences identified with putative roles in pathogenesis included avirulence homologs possessing the RxLR motif, elicitins, and hydrolytic enzymes. This large collection of P. sojae ESTs will serve as a valuable public genomic resource.     

Assessment of gene expression profiles in primary and secondary zoospores of Plasmodiophora brassicae by dot blot and real-time PCR

Information on the molecular basis of pathogenicity of the clubroot pathogen Plasmodiophora brassicae is very limited. Although the sequences of more than 100 P. brassicae genes are available in GenBank, their expression and regulation are largely unknown. In this study, specific primers were designed and used to amplify genomic fragments of 118 P. brassicae genes that represent all database-available proteins and ESTs. The PCR products were blotted on membranes and hybridized with digoxigenin-labeled double-stranded cDNA, derived from either primary or secondary zoospores of P. brassicae. The same primers were also used in real-time PCR against the single-stranded cDNA synthesized from the two types of zoospores. Both dot blot and real-time PCR identified up- and down-regulated genes and the correlation between these two techniques was confirmed. Real-time PCR indicated that 58 genes were up-regulated in the secondary zoospores relative to the primary zoospores, whereas 55 were down-regulated. These data suggest that different mechanisms are utilized by the pathogen in causing primary and secondary infections. The expression patterns of genes with known or putative functions suggest the relative importance of these genes during pathogenesis. In contrast, highly expressed or regulated genes with unknown function can be further studied in identifying pathogenicity factors.     

Identification of Alternaria brassicicola genes expressed in planta during pathogenesis of Arabidopsis thaliana

Alternaria brassicicola is a necrotrophic fungal pathogen that causes black spot disease on cruciferous plants including economically important Brassica species. The purpose of this study was to identify fungal genes expressed during infection of Arabidopsis. In order to identify candidate genes involved in pathogenicity, we employed suppression subtractive hybridization (SSH) between RNA isolated from A. brassicicola spores incubated in water and on the leaf surface of the Arabidopsis ecotype Landsberg. Two populations of cDNA were created from total RNA extracted after 24h when approximately 80% of the spores had germinated either on the leaf surface or in water. Following SSH, expression of clones was examined using dot-blot macro-arrays and virtual Northern blots. 47 cDNA clones differentially expressed between Alternaria infected Arabidopsis leaves and spore germination in water were selected for sequencing. Seventy-seven percent (36) of the cDNAs had significant homology to fungal sequences from databases examined, including available fungal genomes, while 13% (11) had no homology to sequences in the databases. All 36 genes had significant matches with genes of fungal origin, while 11 genes did not have significant hits in the databases examined. Five sequences were expressed on the plant leaf surface but not during spore germination in water according to virtual Northern blots. These five cDNAs were predicted to encode a cyanide hydratase, arsenic ATPase, formate dehydrogenase, major Alternaria allergen, and one unknown. RT-PCR was used to examine the expression of these five genes during infection of Brassica oleraceae var. capitata (cabbage), in vitro growth in nutrient rich media, and infection of Arabidopsis thaliana. Four of these genes are expressed in the nutrient rich medium, while the unknown gene P3F2 was only expressed during plant infection. The results of this study provide the first insight into genes expressed during A. brassicicola infection of Brassica species that may be involved in fungal pathogenesis.