Translocation of the fungal effector AvrM into host cells

Abstract

Rust fungi are obligate biotrophic pathogens that are responsible for some of the most devastating diseases of economically important plants. It is thought that these pathogens are able to evade host defense responses and obtain nutrients from living host tissue through the actions of small, secreted proteins known as effectors, which alter the structure and function of plant cells. Therefore, elucidating the fate of these proteins during infection is important for understanding the molecular mechanisms underlying infection and for development of better disease control measures. The experiments described in this thesis aim to address this issue by examining the localisation of the flax rust effector AvrM during infection by the fungus of the host plant and by exploring molecular mechanisms potentially responsible for AvrM uptake into plant cells. Two approaches were taken in the localisation studies: (1) immunolocalisation of AvrM and (2) the generation of transgenic flax rust fungi expressing AvrM-fluorescent protein fusions. Although flax rust transformants expressing AvrM fusion proteins were successfully isolated and fusion proteins could be detected on immunoblots, no fluorescent signal was observed. Thus, AvrM distribution was studied primarily by immunolocalisation. For this purpose, polyclonal antibodies against AvrM were generated and used in immunofluorescence and immunogold labelling experiments. While the antibodies did not detect the presence of AvrM on spores or in vitro germinated spores, labelling was observed in planta at the periphery of fungal hyphae and specialised fungal feeding structures known as haustoria. Strong cytoplasmic labelling was also detected within the cytoplasm of infected plant cells providing direct evidence that AvrM enters the host cell during infection. Examination at the electron microscope level indicated the presence of the protein in vesicles close to the host-derived extrahaustorial membrane at the plant-pathogen interface. Labelling was also associated with putative endomembrane structures within host cells. Having confirmed the transfer of AvrM into the host during infection, the mechanism of effector uptake into host cells was investigated. This involved using viral-induced gene silencing of plant genes implicated in clathrin-mediated endocytosis and clathrin-independent endocytosis as well as the expression of dominant negative AP180 homologs, which are predicted to affect clathrin-mediated endocytosis. The results of these experiments did not provide conclusive evidence for the involvement of any of the tested pathways in AvrM uptake. Finally, in light of evidence that lipid binding may be required for effector uptake into host cells, the lipid binding activities of AvrM and AvrL567, another flax rust effector, were tested using various deletion and truncation mutants of both proteins. This showed that while AvrL567 did not possess significant lipid binding activity, AvrM bound strongly to phosphatidylinositol, phosphatidylinositol monophosphates and phosphatidyl serine. However, two AvrM truncations that were able to enter host cells in planta displayed loss of lipid binding activity, suggesting that lipid binding is not required for uptake. The significance of the results obtained in the present study as well as future directions for research are discussed. Show more