Characterisation of flagellar mastigoneme components of phytophthora nicotianae

Abstract

Plant diseases caused by Phytophthora species pose significant threats to agriculture and natural biodiversity throughout the world. Motile, flagellate zoospores of Phytophthora and most Oomycete species play a key role in pathogen dissemination and initiation of infection of host plants. Tripartite tubular hairs called mastigonemes on the zoospore anterior flagellum are responsible for cell motility by reversing the thrust of flagellar beat. The objective of the work presented in this thesis was to identify and characterise components of Phytophthora mastigonemes. In previous studies, peptide sequencing and electron microscopy using an antibody directed towards mastigonemes of Phytophthora nicotianae zoospores had identified a gene, PnMas2, which encodes a protein in the tubular shaft of the mastigoneme. Bioinformatic analysis revealed that PnMas2 belongs to a protein family consisting of three or four members. In the current study, degenerate primers were used to clone PnMasl and PnMas3, the two other members of the Mas family in the P. nicotianae genome. Homologues of the three PnMas genes were identified in the genomes of 30 species of Stramenopiles, the major protist assemblage to which the Oomycetes belong. Phylogenetic analyses indicated that the Mas family can be divided into three sub-families, namely Masl, Mas2 and Mas3. Extensive BLAST analyses of Stramenopile and other eukaryotic genomes demonstrated that the Mas protein family occurs only within the Stramenopile taxon and is not found in those few Stramenopile species that do not produce flagellate cells. Quantitative real-time PCR showed that all three Mas genes are expressed during asexual sporulation but that relative levels of expression at other stages of the asexual life cycle differ for the three genes. This suggests that the Mas proteins may have different functions apart from their role in flagellar mastigonemes. Monoclonal antibodies (mAbs) specific for PnMasl and PnMas3 were generated to study the localisations and protein-protein interactions of the two putative mastigoneme proteins. On immunoblots, anti-PnMasl and anti-PnMas3 each reacted with a single polypeptide with a relative molecular weight close to that predicted from the amino acid sequence of the protein - 64 kDa for PnMasl and 24 kDa for PnMas3. Immunoblotting of zoospore protein extracts in native gels indicated that PnMasl and PnMas2 both PnMas proteins has provided clues as to their function. The new information resulting from my study will contribute to increase the understanding of Phytophthora zoospore motility and subsequently the mechanism of Oomycete disease dissemination. Show more