Identification of mechanisms defining resistance and susceptibility of Camellia plants to necrotrophic petal blight disease : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Manawatū, New Zealand

Abstract

Listed in 2019 Dean's List of Exceptional ThesesAll Figures are re-used with publishers' permission.Species in the genus Camellia, which includes the tea crops, oil-producers and valuable ornamental plants, have economic and cultural significance for many countries. The fungus Ciborinia camelliae causes petal blight disease of Camellia plants, which has a short initial asymptomatic phase and results in rapid necrosis and fall of blooms. Ciborinia camelliae is a necrotrophic pathogen of the family Sclerotiniaceae, which also includes two broad-host range necrotrophic pathogens, Botrytis cinerea and Sclerotinia sclerotiorum. Previously it was shown that some Camellia plants, such as Camellia lutchuensis, are naturally resistant to petal blight. In order to find molecular mechanisms underpinning this resistance, a genome-wide analysis of gene expression in C. lutchuensis petals was conducted. The analysis revealed a fast modulation of host transcriptional activity in response to C. camelliae ascospores. Interaction network analysis of fungus-responsive genes showed that petal blight resistance includes increased expression of important plant defence pathways, such as WRKY33-MPK3, phenylpropanoid and jasmonate biosynthesis. A much-delayed activation of the same pathways was observed in the susceptible Camellia cultivar, Camellia ‘Nicky Crisp’ (Camellia japonica x Camellia pitardii var. pitardii), suggesting that failure to activate early defence enables C. camelliae to invade and cause tissue necrosis. Early artificial induction of defence pathways using methyl jasmonate reduced the rate of petal blight in susceptible ‘Nicky Crisp’ plants, further verifying the role of a rapid defence activation in petal-blight resistance. Overall, transcriptomic and functional analysis of the Camellia spp.- C. camelliae interaction demonstrated that the same plant defence pathways contribute to both resistance and susceptibility against this necrotrophic pathogen, depending on the timing of their activation. To further understand the molecular mechanisms of petal blight resistance, the role of the phenylpropanoid pathway, identified as a key feature in the transcriptome study above, was investigated in more detail. This pathway produces various metabolites, including phenolic acids, aldehydes, and alcohols, which have numerous physiological functions and also participate in the production of flavonoids and lignin. Resistant C. lutchuensis was shown to rapidly activate the expression of core phenylpropanoid genes after treatment with C. camelliae ascospores. LC-MS-based quantification of phenylpropanoid compounds demonstrated that within the first 6 h of the infection, resistant plants had already accumulated coumaric, ferulic and sinapic acids, while at 24 hpi, concentrations of coumaraldehyde, sinapaldehyde, and caffeyalcohol were significantly increased. Thus, I further hypothesized that the compounds produced by the phenylpropanoid pathway may have fungistatic activity. Indeed, all tested phenylpropanoids inhibited the growth of C. camelliae in agar plates with different efficacy. Moreover, the application of phenylpropanoid compounds, including ferulic and coumaric acids, fully prevented the formation of petal blight lesions on susceptible Camellia ‘Nicky Crisp’ petals. Taken together, it can be concluded that the phenylpropanoid pathway may contribute to the early defence against the petal blight via the rapid production of fungistatic compounds. Ultimately, these compounds could be used to develop natural antifungal sprays to protect susceptible Camellia flowers. The analysis of the C. camelliae secretome using LC-MS/MS detection of proteins showed that the pathogen produces a large number of carbohydrate-active enzymes in liquid culture and plant petals. Injection of these proteins induced necrosis not only in susceptible Camellia petals but also in petals of the resistant species and leaves of non-host Nicotiana benthamiana. It was proposed that these enzymes can contribute to the virulence of the pathogen by inducing cell death and facilitating necrosis propagation. Thus, the early defence responses of resistant Camellia plants may possibly stop the development of C. camelliae before it starts releasing carbohydrate-active enzymes during the necrotrophic step of the infection. Overall, the results of this research further expand our understanding of plant- necrotroph interactions, suggesting that the timing of plant immune responses may be a crucial factor defining the outcome of the necrotrophic infection