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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Date
2019
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Massey University
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Abstract
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.
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Keywords
Pathogenic fungi, Molecular aspects, Camellias, Diseases and pests, Botrytis cinerea, Sclerotinia sclerotiorum, Cell death