The Phytophthora nicotianae zoospore secretome

Abstract

Plants and their pathogens are continually co-evolving in what has been referred to as an evolutionary arms race. In this struggle between pathogen and host, the adaptation of one can lead to a selective pressure and in turn a counter-adaptation of the other. The selective pressure on secreted pathogen proteins is particularly high as these proteins have functions vital to successful infection and maintaining a parasitic relationship. In this thesis a proteomics approach was taken to investigate the proteins secreted during the initial stages of infection of Phytophthora nicotianae. This pathogen belongs to the Oomycetes, which includes many destructive plant pathogenic species. The diseases caused by these species affect many different plant hosts and impact not only agricultural production but also native ecosystems. During the initial stages of plant infection by P. nicotianae, motile zoospores locate an appropriate infection site and undergo encystment where they detach both flagella, form a cell wall and secrete proteins from three types of peripheral vesicles found in the zoospore cortex. In Chapter 2, the timing of synthesis of four proteins (Cpa, Lpv, PnCcp and Vsv) found in these vesicles was investigated during asexual sporulation. The results showed differences in the time at which Cpa, Lpv, PnCcp and Vsv were synthesised. In addition, the timing of expression of Lpv, PnCcp and Vsv genes was examined in sporulating hyphae, zoospores and 3h germinated cysts. It was found that these genes had different expression patterns. In preparation for the identification and characterisation of P. nicotianae transformants in which one of these secreted proteins, namely PnCcp, was silenced, assays for screening and analysing transformants were developed. While sequence motifs in PnCcp and Vsv indicate that these two proteins are likely to have adhesive properties, their function has not been directly demonstrated. In Chapter 3, an RNAi gene silencing approach was used to investigate the function of PnCcp. Chapter 4 focused on an investigation of protein secretion during zoospore encystment. Firstly, spatial and temporal patterns of secretion of the four peripheral vesicle proteins were determined. Lpv was found not to be secreted and there were differences in the timing of secretion of the other three proteins with Cpa being secreted earlier than Vsv and PnCcp. Secondly, proteomic and bioinformatic approaches were used to investigate proteins secreted during zoospore encystment. This analysis led to the identification of a number of proteins in extracts solubilised from encysted zoospores and 29 of these were predicted to have N-terminal secretion signals by SignalP. The majority of these proteins had unknown functions, highlighting the need for more research in this area. Other putative secreted proteins were involved in carbohydrate and protein metabolism. Together, the research reported in this thesis sheds new light on the synthesis and secretion of zoospore vesicle proteins and provides a foundation for further research aimed at characterising the function of these proteins