Gene expression data analysis using novel methods: Predicting time delayed correlations and evolutionarily conserved functional modules

Microarray technology enables the study of gene expression on a large scale. One of the main challenges has been to devise methods to cluster genes that share similar expression profiles. In gene expression time courses, a particular gene may encode transcription factor and thus controlling several genes downstream; in this case, the gene expression profiles may be staggered, indicating a time-delayed response in transcription of the later genes. The standard clustering algorithms consider gene expression profiles in a global way, thus often ignoring such local time-delayed correlations. We have developed novel methods to capture time-delayed correlations between expression profiles: (1) A method using dynamic programming and (2) CLARITY, an algorithm that uses a local shape based similarity measure to predict time-delayed correlations and local correlations. We used CLARITY on a dataset describing the change in gene expression during the mitotic cell cycle in Saccharomyces cerevisiae. The obtained clusters were significantly enriched with genes that share similar functions, reflecting the fact that genes with a similar function are often co-regulated and thus co-expressed. Time-shifted as well as local correlations could also be predicted using CLARITY. In datasets, where the expression profiles of independent experiments are compared, the standard clustering algorithms often cluster according to all conditions, considering all genes. This increases the background noise and can lead to the missing of genes that change the expression only under particular conditions. We have employed a genetic algorithm based module predictor that is capable to identify group of genes that change their expression only in a subset of conditions. With the aim of supplementing the Ustilago maydis genome annotation, we have used the module prediction algorithm on various independent datasets from Ustilago maydis. The predicted modules were cross-referenced in various Saccharomyces cerevisiae datasets to check its evolutionarily conservation between these two organisms. The key contributions of this thesis are novel methods that explore biological information from DNA microarray data

Creating novel specificities in a fungal nonself recognition system by single step homologous recombination events

In many organisms, two component systems have evolved to discriminate self from nonself. While the molecular function of the two components has been elucidated in several systems, the evolutionary events leading to the large number of different specificities for self–nonself recognition found in most systems remain obscure. We have investigated the variation within a multiallelic nonself recognition system in the phytopathogenic basidiomycete Ustilago maydis by means of sequence analysis and functional studies. The multiallelic b mating type locus of U. maydis ensures outbreeding during sexual development. Nonself recognition is specified by the two homeodomain proteins, bE and bW, encoded by the b locus. While bE–bW combinations from the same allele do not dimerize, bE and bW proteins originating from different alleles form a heterodimeric complex that functions as master regulator for sexual and pathogenic development. We show that novel specificities of the b mating type locus have arisen by single homologous recombination events between distinct b alleles that lead to a simultaneous exchange of subdomains involved in dimerization in both bE and bW, altering the specificity of both proteins in a single step

Multiallelic recognition: Nonself-dependent dimerization of the bE and bW homeodomain proteins in ustilago maydis

AbstractIn the plant pathogenic fungus Ustilago maydis, sexual and pathogenic development are controlled by the multiallelic b mating-type locus. The b locus encodes a pair of unrelated homeodomain proteins termed bE and bW, with allelic differences clustering in the N-terminal domains of both polypeptides. Only combinations of bE and bW of different allelic origin are active. We have investigated the underlying molecular mechanism for this intracellular self/nonself recognition phenomenon. By using the two-hybrid system, we were able to show that bE and bW dimerize only if they are derived from different alleles. Dimerization involves the N-terminal variable domains. Different point mutants of bE2 were isolated that function in combination with bW2. The majority of such bE2 mutant polypeptides were also able to form heterodimers with bW2 in the two-hybrid system. Nonself-dependent dimerization of bE and bW was supported with a biochemical interaction assay with immobilized proteins. Our results suggest a model for self/nonself recognition in which variable cohesive contacts direct dimerization

The Nma1 protein promotes long distance transport mediated by early endosomes in Ustilago maydis

Early endosomes (EEs) are part of the endocytic transport pathway and resemble the earliest class of transport vesicles between the internalization of extracellular material, their cellular distribution or vacuolar degradation. In filamentous fungi, EEs fulfill important functions in long distance transport of cargoes as mRNAs, ribosomes, and peroxisomes. Formation and maturation of early endosomes is controlled by the specific membrane-bound Rab-GTPase Rab5 and tethering complexes as CORVET (class C core vacuole/endosome tethering). In the basidiomycete Ustilago maydis, Rab5a is the prominent GTPase to recruit CORVET to EEs; in rab5a deletion strains, this function is maintained by the second EE-associated GTPase Rab5b. The tethering- and core-subunits of CORVET are essential, buttressing a central role for EE transport in U. maydis. The function of EEs in long distance transport is supported by the Nma1 protein that interacts with the Vps3 subunit of CORVET. The interaction stabilizes the binding of Vps3 to the CORVET core complex that is recruited to Rab5a via Vps8. Deletion of nma1 leads to a significantly reduced number of EEs, and an increased conversion rate of EEs to late endosomes. Thus, Nma1 modulates the lifespan of EEs to ensure their availability for the various long distance transport processes

Robust Cre recombinase activity in the biotrophic smut fungus Ustilago maydis enables efficient conditional null mutants in planta

Site-specific recombinases have been used in higher eukaryotes, especially in animals, for a broad range of applications, including chromosomal translocations, large deletions, site-specific integration, and tissue-specific as well as conditional knock-outs. The application of site-specific recombination has also been demonstrated in simple eukaryotes like fungi and protozoa. However, its use in fungal research, especially in phytopathogenic fungi, has often been limited to “recycle” the marker genes used in transformation experiments. We show that Cre recombinase can be used for conditional gene deletions in the phytopathogenic fungus Ustilago maydis. Conditional gene knock-outs can be generated via the transcriptional control of the recombinase by U. maydis promoters specifically activated during the biotrophic phase of fungal growth, enabling gene deletions at defined developmental stages inside the plant tissue. Also, we show that a tamoxifen-activated Cre-recombinase allows the tight control necessary for the induced deletion of essential genes by the addition of tamoxifen. These tools will be helpful to address the function of genes under both axenic and in planta conditions for the U. maydis-maize pathosystem and should pave the way for similar approaches in other plant pathosystems

Basidiomycete Genomics

Non peer reviewe

Cytoplasmic Transport Machinery of the SPF27 Homologue Num1 in Ustilago maydis

In the phytopathogenic basidiomycete Ustilago maydis, the Num1 protein has a pivotal function in hyphal morphogenesis. Num1 functions as a core component of the spliceosome-associated Prp19/CDC5 complex (NTC). The interaction of Num1 with the kinesin motor Kin1 suggests a connection between a component of the splicing machinery and cytoplasmic trafficking processes. Previously it was shown that Num1 localizes predominantly in the nucleus; however, due to the diffraction-limited spatial resolution of conventional optical microscopy, it was not possible to attribute the localization to specific structures within the cytoplasm. We have now employed super-resolution localization microscopy to visualize Num1 in the cytoplasm by fusing it to a tandem dimeric Eos fluorescent protein (tdEosFP). The Num1 protein is localized within the cytoplasm with an enhanced density in the vicinity of microtubules. Num1 movement is found predominantly close to the nucleus. Movement is dependent on its interaction partner Kin1, but independent of Kin3. Our results provide strong evidence that, in addition to its involvement in splicing in the nucleus, Num1 has an additional functional role in the cytosol connected to the Kin1 motor protein