Genes Expressed during the Biotrophic Phase of the Rust Fungus Uromyces fabae

Abstract

The subject of this thesis is the study of genes expressed during the biotrophic phase of the rust fungus Uromyces fabae, an obligate biotrophic pathogen of Vicia faba (broad bean).As a first step, a previously initiated partial Expressed Sequence Tag (EST) sequencingproject was completed and the results were analyzed. The aim of this project was to sequence at least 1 000 ESTs and to compare them to publicly available sequences. 58% of the 1 000 plus sequences analyzed using the BLASTX algorithm showed no significant homology to genes published in the NCBI database. The remaining 42% could be classified by functional groups, whereby a surprising number of ESTs bearing similarities to viral sequences were also found.In a second step, three previously identified in Planta Induced Genes (PIGs) were to becharacterized in more detail. This entailed: the internet-based analysis of unusual sequence characteristics; the determination of the number of gene copies; the localization of gene product in infected plant tissue; the verification of in planta gene expression; and the verification of the secretion signal.To this end, six promising PIGs from the above group of ESTs were selected based on two important common characteristics: not only had an internet-based analysis revealed them to possess a secretion signal, but they also displayed no or very minor homology to publicly available sequences at the NCBI database.The number of gene copies for each of the six PIGs was determined by Southern blotting,with the following results: PIG14, PIG15 and PIG23: one copy each; PIG5: two copies; PIG7 (Rust Transferred Protein 1, RTP1): presumably two copies with a single nucleotidepolymorphism (SNP); PIG9: one to two copies.Subsequently, an attempt was made to localize the gene products within the infected plant tissue by using antibodies against the PIG-proteins being studied. In order to produce antibodies, fusion proteins of the six PIGs were over-expressed in E. coli. During the subsequent protein purification process, the fusion proteins of PIG9 and PIG23 proved to be unstable. Thus, PIG9 and PIG23 were not studied any further. Using immuno-fluorescence microscopy, it was shown that the gene product of PIG5 is located inside the haustoria and the gene products of PIG14 and PIG15 are located in the extrahaustorial matrix. Most intriguingly, the gene product of PIG7 (RTP1) was found in the extrahaustorial matrix as well as in the plant(!) nucleus. This result correlated very well with the nuclear localization signals (NLS) found in PIG7p through an internet-based analysis.Finally, the in planta expression of PIG5, PIG7 (RTP1), PIG14 and PIG15 was verified byNorthern blotting. The PIG-proteins were additionally expressed in transformed yeasts (S. cerevisiae), thus allowing the secretion signals to be positively verified for PIG7p and PIG14p using Western blotting.The localization of PIG14p and PIG15p in the extrahaustorial matrix as well as thelocalization of PIG7p (RTP1p) in the extrahaustorial matrix, parts of the host cytoplasm and the plant nucleus provides supporting evidence that these proteins, especially RTP1p, play a (significant?) role in the interactions between U. fabae and V. faba during the biotrophic phase. Furthermore, the fact that PIG7p (RTP1p) can be found in the plant nucleus could be an indication that this protein is involved in the establishing of plant fungal interactions.