Elucidating Genetic Variation and Mechanism of Virus Infection of Emiliania huxleyi via Genomic Approaches

Abstract

This thesis aimed at the identification of genes involved in the host-virus interaction of the coccolithophore Emiliania huxleyi and the virus EhV-86 by taking advantage of EST libraries and DNA microarrays. Microarray-based comparative genomic hybridization (CGH) was used to investigate the genetic variation of several E. huxleyi strains and to identify genes with respect to virus susceptibility and morphology, e.g. formation of coccoliths.Analysis of expressed sequence tags (ESTs) was performed to gain insights into the host-virus interaction of E. huxleyi. Three complementary DNA (cDNA) libraries generated 6, 12 and 24 h post viral infection were compared to a library from an uninfected culture by sequencing, clustering and manual annotation of randomly selected ESTs. At first, a preliminary set of 60-90 ESTs from each library were annotated to get an overview of gene expression changes that occur during viral infection of E. huxleyi. BLAST-searches of the sequenced genome of the virus (EhV-86) were used to identify viral genes. Results of this small sample probe show already a trend towards down-regulation of genes involved in photosynthesis of E. huxleyi for the benefit of an increased transcription and translation for viral replication. At 6 (T6) and 12 (T12) hours post viral infection the algal transcriptome changed significantly although only 3-4 viral transcripts were present. In addition, at 24 hours (T24) post infection only 10% of the mRNA was of host origin. Viral transcripts identified at T24 encode proteins involved in protein degradation, nucleic acid degradation, transcription and replication.As a next step, 1100-1500 ESTs per library were sequenced and annotated. Results confirmed the previous tendencies and discovered more genes involved in the host's response to viral infection. Furthermore, two-colour oligonucleotide microarrays were used to verify the gene expression results of the ESTs. A total of 4480 ESTs were assembled into 1871 clusters of which 223 clusters were of viral origin. A putative function could be assigned to 35% of the host clusters and to 20% of the viral clusters.In addition, microarray expression analysis indicated that 231 out of 565 oligonucleotides of E. huxleyi changed their expression level in at least one time point. Results suggest that viral infection affects the transcriptional machinery of E. huxleyi within a few hours by decreasing the expression of genes involved in photosynthesis and protein degradation at the benefit of fatty acid metabolism, glycolysis, and transcription and translation. The expression of E. huxleyi and EhV-86 genes changed significantly between 12 and 24 hours after infection.The results provide insights into the infection mechanisms of the virus EhV-86 in E. huxleyi and demonstrate the power of EST libraries and DNA microarrays to obtain data on gene expression and regulation during viral infection.Microarray-based comparative genomic hybridization (CGH) was applied to investigate genomic diversity of 16 E. huxleyi strains of different geographic origin and to identify genes related to virus susceptibility and morphology. The microarray consisted of 565 genes derived from the former EST study of E. huxleyi strain CCMP1516 and 37880 genes from the ongoing genome project of the same strain. Gephyrocapsa oceanica and Isochrysis galbana were taken as out-groups. A total of 32395 gene transcripts showed significant hybridization patterns and were used to elucidate genetic diversity. Hybridization intensities were compared to determine the relative copy number of each gene transcript. Comparisons with the sequenced E. huxleyi strain CCMP1516 revealed that 27% (8740 genes) to 57% (18581 genes) of the genes showed a pattern of hybridization concordant with deletion, nucleotide divergence or gene duplication within the species and up to 83% (26881 genes) between the genera. The largest variation was observed among the species for E. huxleyi strain 92F. Regarding variation with respect to virus susceptibility and morphology the most abundant genes with known function were associated with metabolism, transport, and transcription and translation. In addition, two membrane receptors and two proteins related to ubiquitin were identified which show significant differences between virus susceptible and resistant strains.The results obtained by using CGH demonstrate that this method is appropriate to compare the gene content of different E. huxleyi strains. CGH was successfully applied to identify genes related to virus susceptibility and morphology. Among others, the membrane receptors and the ubiquitin-related proteins that possibly play a role in virus infection deserve further attention.