| Abstract |
A diverse group of fungi and oomycetes, including members of the genera Phytophthora and Colletotrichum spp., have pathogenic lifestyles on the plants. Elucidation of disease mechanisms for such microorganisms is often hampered by lack of suitable genetic analysis tools. RNAi (RNA-mediated gene silencing) allows for the targeted inhibition of gene expression at the post-transcriptional level (gene “knockdown”). This method constitutes the most recent development in the repertoire of genetic analysis tools for eukaryotic organisms and is being increasingly employed in the investigation of host-pathogen interactions involving plants and lower eukaryotic pathogens. This dissertation describes preliminary efforts to establish
RNAi-mediated methodologies in a plant pathogenic ascomycete (Colletotrichum higginsianum) and two oomycete phytopathogens (Phytophthora parasitica and Aphanomyces euteiches).
In order to develop and optimize the RNAi methodology we initially chose the sgfp gene as a suitable marker gene to be targeted. The strategy which was followed was to firstly introduce the gene into the organism under study and subsequently to trigger its silencing by introducing specially designed sGfp-specific silencing cassettes. To implement this strategy, we designed primers with EcoRI restriction
sites and amplified a 330 nt sequence from the sGfp gene variant, cloned this fragment into the pENTRY3C vector (GATEWAY SYSTEM) in both directions, and, by applying two sequential LR reactions, one for each insert direction, we transferred them to the destination vector pK7GWIWG2(I),0, thus, constructing two hairpin cassettes (As/S and S/As). These hairpin cassettes were then transferred to two expression vectors, pNC2 for Phytophthora and the binary agrobacterium vector pBIG4MRBrev for Colletotrichum. A series of additional sGfp cassettes (sense and antisense) for stable transformation were also designed for the generation of sGfp
expressing transformants and for silencing of the sGfp transgene. The hairpin and antisense cassettes were expected to generate double-stranded RNA molecules and eventually trigger the silencing of the sGfp marker gene. By applying fungal protoplast transformation and Agrobacterium tumefaciens-mediated transformation, we attempted to induce the sGfp and silencing constructs. Putative positive transformants were obtained by selection for antibiotic resistance on appropriately supplemented agar plates and were examined for the presence of green fluorescent
mycelium by fluorescence microscopy. Efficient RNAi methodology would further enable the study of the biological role of putative fungal phytopathogenesis proteins (effectors) and genes such as those
involved in cell wall biosynthesis. The cell walls of oomycetes were generally considered to be devoid of chitin. However, recent evidence indicated the presence of chitin as well as of putative chitin biosynthesis genes (Chs) in P. parasitica and A. euteiches. Accordingly, we sought to isolate Chs genes from P. parasitica and A.
euteiches. Moreover, we sought to isolate Chs genes as a prelude to designing constructs for Chs gene silencing in these fungi. Two full-length Chs cDNAs were isolated from A. euteiches (encoding ΑeCHS 1 and AeCHS 2) and one partial-length Chs cDNA from P. parasitica (Ppn0_Chs) coding for the more conserved part of the
protein. Phylogenetic analysis showed that the Chs genes in these oomycetes arose from duplication of an ancestral gene which gave origin to two distinct clades for Chs among phytopathogenic oomycetes. Based on the above, a silencing antisense cassette
was designed for the Ppn0_Chs gene.
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Μετά-μεταγραφική γονιδιακή σίγηση σε μύκητες και ωομύκητες
