Elucidating the molecular basis pf Phytophthora zoospore motility: identifying the protein compostion of flagellar hairs

A project undertaken at Plant Sciencises Division, Research School of Biology, Australian National University, and supervised by Adrienne Hardham

Phytophthora species are fungus-like plant pathogens that cause devastating diseases in important crops and native plants.   In Australia, Phytophthora diseases lead to extensive economic losses in agriculture, horticulture and forestry and are a major threat to natural ecosystems and biodiversity.   Phytophthora species are protists phylogenetically related to diatoms, coloured algae and malarial parasites.   In general, Phytophthora pathogens are not effectively controlled by traditional fungicides and new, safe and sustainable control strategies are urgently needed.   This project aimed to address this need by increasing our understanding of the cellular and molecular basis of the infection process.

One of the distinctive features of Phytophthora species is their production of motile biflagellate zoospores.   These zoospores are able to swim chemotactically to suitable infection sites on potential host plants.   The ability of zoospores to swim to host plants is a key aspect of successful initiation of infection.   Zoospore motility is achieved through the action of tripartite tubular hairs, called mastigonemes, on the anterior flagellum.   One proteinaceous mastigoneme component, PnMas2, had been cloned and shown to be part of the tubular shaft of the hairs.   Bioinformatic analysis indicated that PnMas2 was a member of a small multi-gene family.  The main goal of this project was to identify other components of the mastigonemes with a view to increasing our understanding of their structure and function.

During the course of this research, we cloned and characterised the other two members of the Mas gene family in Phytophthora nicotianae.  All three genes have a signal peptide directing protein secretion and contain four epidermal growth factor-like domains.  These are likely to be involved in protein-protein interactions.  Quantitative real-time PCR was used to determine expression profiles for the three PnMas genes.  The results showed that while the three genes were all strongly expressed during sporulation, when most zoospore components are synthesised, at other stages of the asexual life cycle, the genes showed different patterns of expression.  This result suggests that the three proteins may play distinct functions.

Antibodies specific for PnMas1 and PnMas3 were generated and used in immunoblot and immunocytochemical labelling experiments.  Anti-PnMas1 antibodies showed that this 64 kDa protein formed part of the mastigoneme shaft.  Co-immunoprecipation experiments and immunolabelling of blots from native gels provided evidence that PnMas1 and PnMas2 interact in two high molecular weight protein complexes.  Immunolabelling of blots from native gels showed that PnMas3 (Mr 24 kDa) did not interact with PnMas1 or PnMas2 but was part of a 60 kDa protein complex.  Transformants expressing a PnMas3-GFP fusion protein showed the presence of PnMas3 within the ER but not associated with mature mastigonemes on the flagellar surface.  This result suggests that PnMas3 may be involved in mastigoneme assembly but not in mastigoneme function.  Work is currently underway to further examine this possibility.

In summary, the research conducted with support from the Herman Slade Foundation has allowed us to expand our understanding of the structure of Phytophthora mastigonemes.  The new knowledge gained will be important for studies of the structure and function of the flagellar apparatus in this and other Stramenopile organisms.  In the case of pathogenic species, our research may provide the basis for development of novel control measures that inhibit spore motility and the initiation of disease. 



Figure 1. Mastigonemes (green) are rigid, tripartite hairs that are arranged in two opposing rows along the anterior flagellum of biflagellate, Stramenopile zoospores. They are visualised in this immunofluorescence image of a Phytophthora zoospore by labelling with an antibody that reacts with a proteinaceous component of the shaft of the tripartite mastigonemes.