Browsing by discipline "Biotechnology (METSÄ)"

Phytopathogens, notably Heterobasidion annosum, evolved several strategical combinations to infect and subsequently colonize their host even under different stress conditions. Fungal ABC transporters are well-known defenses that can confer resistance against host-secreted secondary metabolites by transporting them outside of the fungal cells and thus keeping their intracellular concentration low. Here, we aim to unveil the evolutionary trajectories of total ABC transporters-encoding genes in Heterobasidion annosum. The gene expression pattern was monitored with the fungus subjected to different chemical stressors and during fungal growth on wood. We identified 32 putative ABC protein-encoding genes in the Heterobasidion genome. Altogether 20 putative ABC transporter-encoding genes of H. annosum were further analyzed and it was revealed that several genes were either up or downregulated, while some were not differentially expressed under the experimental conditions. The results obtained from the gene expression analysis revealed that an ABC gene (annotated as Ha.ABC-G1 or Hetan_66124), was highly up-regulated in most conditions. This particular transporter-encoding gene (Hetan_66124) with induction level of up to 47 –fold (in heartwood and similar levels in other conditions) was traced, PCR amplified, cloned in Escherichia coli and expression of recombinant protein performed using Saccharomyces cerevisiae as platform. Several experiments aiming to dissect functional roles of this hypothetical protein were performed. The growth of the yeast transformant over expressing the recombinant ABC protein in different terpenoids and weak organic acids were monitored. The growth rate of clones with and without transporters were not significantly different when cultured in plates (SC·gal-ura-) that were exposed to the volatile compounds (limonene, carene and ?-pinene). Based on our findings, we concluded that the yeast transformants carrying the H. annosum ABC-G1 transporter encoding gene do not show increased resistance or tolerance against the monoterpenes. The results of the transcript profiling have further contributed to our understanding about gene expression during fungal colonization upon exposure to chemical stressors. However, further studies are needed in order to specifically unveil the functional roles of these efflux pumps that underlie their transport mechanism with response to the host secreted secondary metabolites.