Lu, Yi-Ju (2011) Live-cell imaging reveals subcellular localization of plant membrane compartments during oomycete infections and quantitative high-throughput imaging identifies endocytic trafficking mutants. PhD thesis, MPI fur Züchtungsforschung.
To successfully infect plants, filamentous pathogens such as the oomycete Hyaloperonospora arabidopsidis (Hpa) are able to penetrate host tissues and form haustoria, a feeding structure, inside the host cell. Reorganization of the host cell is required to accommodate the haustoria. Formation of haustoria is accompanied by the biogenesis of the extrahaustorial membrane (EHM) which surrounds the haustorium and separates the host cell from the pathogen. In this study, available fluorescent marker protein fusions were used to monitor the re-distribution of membrane compartments at the interface between Arabidopsis and Hpa. The aquaporin PIP1;4, the ATPase ACA8, and the plasma membrane (PM) intrinsic protein NPSN12 were excluded from the EHM while the syntaxin PEN1 and the receptor-like kinase FLS2 labelled the EHM. This suggests PM-resident proteins are recruited to the EHM selectively. The nucleus is always observed adjacent to haustoria, suggesting that the presence of haustoria causes migration of the nucleus. Secretory vesicles and endosomal compartments localize around the haustoria, implicating secretory and endocytic pathways in the biogenesis of the EHM. Upon Hpa infection, haustorial encasements develop around mature haustoria. All examined plant proteins accumulate at haustorial encasements, indicating that formation of encasements is derived by default redirection of vesicle trafficking pathways. With the aim to genetically dissect endosomal trafficking regulators, I took advantage of quantitative high throughput confocal imaging system and transgenic plants containing the fluorescent biosensor GFP-2xFYVE to perform a forward genetic screen. Different numbers of GFP-2xFYVE positive endosomes were found in two reference lines, Ler/GFP-2xFYVE and Col-0/YFP-2xFYVE suggesting the endosomal levels may vary in different ecotypes of Arabidopsis. Mutants with altered numbers of FYVE Endosomal Levels (fel) have been previous identified and were re-confirmed in this study. fel1, fel2, fel3, fel6, fel9, and fel12 revealed genetically recessive mutations while fel10 could not reveal its genetic inheritance. Two mutants, fel2 and fel9 exhibited more GFP-2xFYVE compartments than wild-type reference plants. These two mutants are affected in endosome trafficking and fel2 is likely tissue specific. We identified gene loci by classical mapping and whole genome sequencing. Fel2 co-segregated with the lower arm of chromosome 4. Fel9 was mapped to two chromosome loci. Investigation of genes in the rough mapping region will unravel regulators of endocytosis or multivesicular bodies (MVBs) biogenesis. Because only few mutant phenotypes recovered in the F2 of backcrossed fel2 and fel9, identification of FEL2 and FEL9 was hampered. Additionally, basal differences of endosomal numbers in the reference lines lead to the limitation for genetic screen based on quantitative changes in endosomal numbers. Altogether, these results show that there are common elements in the subcellular changes associated with biotrophic oomycete between different pathogens. For Hpa and other fungal/oomycete pathogens, reprogramming host cell vesicle trafficking occurs to accommodate haustorial structures. A genetic screen for novel endocytosis mutants, based on quantitative measurements of endosomal numbers, was performed with advanced microscopy technology. Fel mutant plants may be further used to study molecular mechanisms for membrane trafficking, as well as subcellular rearrangement in plant-pathogen interactions.
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