Investigation of the Plasmodiophora brassicae life cycle and its interactions with host plants
Plasmodiophora brassicae is a soilborne parasitic protist and the causal agent of clubroot disease in the family Brassicaceae. The formation of characteristic galls on the roots of susceptible hosts disrupts the uptake of water and nutrients by the plant roots, causing stunting, wilting and yellowing of above-ground parts and even premature plant death, resulting in large yield losses and lower oil quality. As an intracellular pathogen, the life cycle of P. brassicae that displays various developmental stages during pathogenesis, is complex. This dissertation describes my efforts during my graduate studies to coordinate the P. brassicae cellular study, investigating the P. brassicae infection process, with the recent published genome sequence and develop tools with which to help in answering the burning question of what happens in resistant cultivars and non-host cultivars, in relation to their susceptible relatives, to prevent cortical infection and terminate disease initiation. I have established a new protocol based on a two-step axenic culture of P. brassicae with its host tissues, for easy and in-planta observation of cellular interactions between P. brassicae and host plants. The double staining of P. brassicae with fluorescent dyes, as established in this study, provides a promising technique for observing the infection process in-planta. Using the live-cell imaging protocols established in the axenic culture, together with transmission electron microscopy, the life cycle of P. brassicae was investigate in its natural pathosystem in soil. P. brassicae infecting a root har or epidermal cell underwent zoosporogenesis between five and 10 days post inoculation (dpi) and completed its life cycle with the production of resting spores, after meiosis and cytoplasmic cleavage of sporulating plasmodia in cortical cells, around 28 dpi. From all the genes involved in meiosis and flagella production, from a diverse array of sequenced eukaryotes, I have identified novel stage specific molecular markers for meiosis and flagella development during the P. brassicae infection cycle. Auxin accumulation in infected root tissues, resulting in cell enlargement to form root galls, could be due to auxin directly secreted by P. brassicae or could be a result of redirected auxin transport towards the site of infection. I carried out a study of the role of the auxin transport system governed by the AUX1 influx protein and the PIN FORMED (PIN)-family efflux proteins during clubroot development in A. thaliana. Transcript analysis of infected tissue showed that host auxin transport was upregulated in early infection stages and down-regulated at the later stages of gall formation. Mutants in auxin eﬂux proteins PIN1 and PIN3 bear smaller galls, suggesting that auxin import into the infected cell is needed for gall formation. Understanding the P. brassicae life cycle and the role of auxin flow in gall development will benefit plant pathologists and plant breeders in developing clubroot disease resistance in canola and other cruciferae vegetables worldwide.
Plasmodiophora brassicae, Life cycle, Arabidopsis, plant-pathogen interaction, auxin transporter
Doctor of Philosophy (Ph.D.)