Barry J. Saville

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.

Differential gene expression in filamentous cells of Ustilago maydis

When fungi interact with plants as pathogens or as symbionts, there are often changes in fungal cell morphology and nuclear state. This study establishes the use of cDNA microarrays to detect gene expression changes in Ustilago maydis cells that differ in structure and nuclear content. Categorizing differentially expressed genes on the basis of function indicated that U. maydis cell types vary most in the expression of genes related to metabolism. We also observed that more genes are up-regulated in the filamentous dikaryon than in the filamentous diploid, relative to non-pathogenic budding cells. Our comparison of pathogenic development indicated that the dikaryon is more virulent than the diploid. Other identified expression patterns suggest a cell-specific difference in nutrient acquisition, cell metabolism and signal transduction. The relevance of gene expression change to cell type biology is discussed.

A comparative genomic analysis of ESTs from Ustilago maydis

A large-scale comparative genomic analysis of unisequence sets obtained from an Ustilago maydis EST collection was performed against publicly available EST and genomic sequence datasets from 21 species. We annotated 70% of the collection based on similarity to known sequences and recognized protein signatures. Distinct grouping of the ESTs, defined by the presence or absence of similar sequences in the species examined, allowed the identification of U. maydis sequences present only (1) in fungal species, (2) in plants but not animals, (3) in animals but not plants, or (4) in all three eukaryotic lineages assessed. We also identified 215 U. maydis genes that are found in the ascomycete but not in the basidiomycete genome sequences searched. Candidate genes were identified for further functional characterization. These include 167 basidiomycete-specific sequences, 58 fungal pathogen-specific sequences (including 37 basidiomycete pathogen-specific sequences), and 18 plant pathogen-specific sequences, as well as two sequences present only in other plant pathogen and plant species.

Contiguous NIF Gene Organization in a Heterocystous Cyanobacterium

Cyanobacteria are a highly diverse, widely distributed group of photosynthetic oxygen-evolving prokaryotes that include some species capable of nitrogen fixation. The diazotrophic members of this assemblage fall into three main categories: the filamentous cyanobacteria, which possess characteristically differentiated nitrogen fixing cells called heterocysts, the filamentous cyanobacteria which lack heterocysts, and the unicellular cyanobacteria. The molecular organization of the nitrogen fixation (nif) genes in these organisms is not yet fully understood. The few reports in the literature of cyanobacteria nif gene arrangement reveal a diversity in the organization of the three nif structural genes, nif K, D and H. (1,2,3,4). Nif K and D code for nitrogenase and nif H codes for nitrogenase reductase. While some cyanobacteria, such as Synechococcus (PCC 7425), have all three genes linked (2), the nitrogenase structural genes of Anabaena sp (PCC 7120) have an 11 kilobase (kb) sequence separating nif K from the contiguous nif DH (1). The removal of this 11 kb sequence and the formation of a contiguous arrangement of nif KDH has been implicated in heterocyst differentiation and nitrogenase expression (5). This study utilized cloned fragments of the Klebsiella and Anabaena 7120 nif structural genes to determine whether or not these two arrangements are found in other cyanobacteria.