Characterization of regulated protein secretion in phytophthora zoospores

Abstract

Secretion of pathogen proteins is crucial for establishment of disease in animals and plants. Typically, early interactions between host and pathogen trigger regulated secretion of pathogenicity factors that function in pathogen adhesion and host penetration. During the onset of plant infection by spores of the Oomycete, Phytophthora nicotianae, proteins are secreted from three types of cortical vesicles. Following induction of spore encystment, two vesicle types undergo full fusion, releasing their entire contents onto the cell surface. However, the third vesicle type, so-called large peripheral vesicles, selectively secretes a small Sushi domain-containing protein, PnCcp, while retaining a large glycoprotein, PnLpv, before moving away from the plasma membrane. The experiments described in this thesis aim to characterize the differential secretion and retention of proteins from the large peripheral vesicles in Phytophthora spores and to investigate the possible mechanisms underlying the selective secretion of large peripheral vesicle proteins. Green fluorescent protein (GFP)-tagged PnCcp proteins were expressed in P. nicotianae by genetic transformation in order to study the subcellular localization and secretion of PnCcp in living cells. Analysis of drug-resistant transformants indicated that sometimes the PnCcp-GFP transgene was not expressed and at other times the fusion protein was not fluorescent or was mislocalized to the cytoplasm or the extracellular space other than the large peripheral vesicles. Double-immunofluorescence labelling of P. nicotianae spores both in vitro and in planta confirmed that PnCcp was secreted from the large peripheral vesicles while PnLpv was retained during encystment. Immunolocalization studies of P. nicotianae mycelia revealed that the synthesis of PnLpv precedes that of PnCcp and PnCcp is added to large peripheral vesicles after PnLpv. By immunofluorescence labelling and enzyme-linked immunosorbent assays (ELISAs), the majority of the PnCcp protein was detected to be secreted within 1-2 min of the onset of encystment and secretion of PnCcp occurred at a similar time period as that of ventral and dorsal vesicle proteins. Selective secretion of PnCcp is associated with its compartmentalization within the vesicle periphery. Pharmacological inhibition of dynamin function, purportedly in vesicle fission, by dynasore treatment provides evidence that selective secretion of PnCcp utilizes a kiss-and-run, transient fusion mechanism similar to that described in animal cells. This is the first report of transient fusion and selective protein secretion outside mammalian cells. Results also show that full or transient fusion of different populations of vesicles is triggered simultaneously by the same signal. The research in this thesis expanded our understanding of regulated protein secretion in Phytophthora and other systems.