Characterisation of the cell death-inducing activity of the conserved family of Ciborinia camelliae-like small secreted proteins (CCL-SSPs) of C. camelliae, Botrytis cinerea and Sclerotinia sclerotiorum : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Manawatu, New Zealand

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Ciborinia camelliae, the causal agent of Camellia petal blight, is a necrotrophic fungus that sequesters nutrients from dead plant cells. Candidate effector proteins have been identified from the secretome as a highly conserved clade, termed C. camelliae-like small secreted proteins (CCL-SSPs). Notably, the CCL-SSPs are not unique to C. camelliae. Indeed, a single homolog of the CCL-SSP family has shown to be encoded by the genomes of the closely related necrotrophs, Botrytis cinerea and Sclerotinia sclerotiorum, (BcSSP and SsSSP, respectively). Previous work has identified the ability of BcSSP and SsSSP to induce cell death on Camellia ‘Nicky Crisp’ petals, whereas of the ten C. camellia CCL-SSPs (CcSSPs) tested, only one induced very weak cell death. The aim of this study was to determine what specific regions of the SsSSP protein confered cell death-inducing ability, and to further characterise the cell death-inducing capability of these CCL-SSPs. In this study it was shown, through generation of chimeric regionswapped proteins and infiltration into Camellia ‘Nicky Crisp’ petals and Nicotiana benthamiana leaves, that the region encoded by Exon 2 of SsSSP is essential for cell death-inducing activity. It was also discovered that BcSSP and SsSSP may induce cell death to different extents, as a significant different was shown in quantified cell death induced on Camellia ‘Nicky Crisp’ petals. It was also found that BcSSP can induce strong cell death on Arabidopsis thaliana leaves, while SsSSP does not. This research also investigated appropriate methods for characterising cell death of CCL-SSPs, and suggested addition of a C-terminus tag for future work. The results of this study have shed further light on the CCL-SSP family as candidate effector proteins and provided several avenues for future researchers to fully elucidate the function of CCL-SSPs and their role in virulence of these three necrotrophic fungi.
Pathogenic fungi, Molecular aspects, Camellias, Diseases and pests, Botrytis cinerea, Sclerotinia sclerotiorum, Cell death