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    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
    (Massey University, 2019) Kondratev, Nikolai
    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.
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    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
    (Massey University, 2019) McCarthy, Hannah
    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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    Characterization of incompatible and compatible Camellia-Ciborinia camelliae plant-pathogen interactions : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2014) Denton-Giles, Matthew
    Many Camellia species and cultivars are susceptible to infection by the host-specific fungal phytopathogen Ciborinia camelliae L. M. Kohn (Sclerotiniaceae). This necrotrophic pathogen specifically infects floral tissue resulting in rapid development of host-cell death and premature flower fall. C. camelliae is considered to be the causal agent of ‘Camellia flower blight’ and is an economically significant pest of both the Camellia floriculture and Camellia oil seed industries. This study sought to identify molecular components that contribute to incompatible and compatible interactions between C. camelliae and Camellia petal tissue. Microscopic analyses of incompatible C. camelliae-Camellia lutchuensis interactions revealed several hallmarks of induced plant resistance, including papillae formation, H2O2 accumulation, and localized cell death. Extension of resistance analyses to an additional 39 Camellia spp. identified variable levels of resistance within the Camellia genus, with Camellia lutchuensis, Camellia transnokoensis and Camellia yuhsienensis exhibiting the strongest resistance phenotypes. Collectively, Camellia species of section Theopsis showed the highest levels of incompatibility. Based on this observation, a total of 18 Camellia interspecific hybrids with section Theopsis species in their parentage were tested for resistance to C. camelliae. The majority of hybrids developed disease symptoms, although the speed and intensity of disease development varied. Hybrids containing high genetic dosages of C. lutchuensis within their parentage were the most effective at resisting C. camelliae infection. Therefore, introgression of genetic information from Camellia lutchuensis into hybrids of Camellia is likely to be a valid approach for breeding Camellia hybrids with increased C. camelliae resistance. A comparison between transcriptomes of mock-infected and infected C. lutchuensis samples identified plant genes that may contribute to C. camelliae resistance, including two putative transcription factors. C. camelliae growth in compatible tissue demonstrated a switch from biotrophy to necrotrophy, evident from the simultaneous development of secondary hyphae and necrotic lesions. The initial biotrophic-like period of C. camelliae growth in planta was asymptomatic; leading to the hypothesis that C. camelliae may secrete fungal effectors during infection. A bioinformatic approach was taken to identify putative effectors of C. camelliae. To facilitate this approach, a 40.7 MB draft genome of C. camelliae was assembled and validated. Genomic and transcriptomic data were used to predict a total of 14711 C. camelliae protein coding genes of which 749 were predicted to form the C. camelliae secretome. The secretome of C. camelliae was compared with the predicted secretomes of the closely related species, Botrytis cinerea (Sclerotiniaceae) and Sclerotinia sclerotiorum (Sclerotinaceae). Comparative analysis of the secretomes of these three species identified protein conservation within CAZyme (carbohydrate active enzyme) and protease categories. In contrast, the oxidoreducatase and small secreted protein (SSP) categories were less conserved. Further analysis of SSPs revealed a conserved family of putative effector proteins that appear to have undergone lineage-specific expansion within the host-specific pathogen C. camelliae (n = 73), but remain as single genes in the two host-generalists B. cinerea and S. sclerotiorum. Members of this Ciborinia camelliae-like small secreted protein (CCL-SSP) family share 10 conserved cysteine residues, are expressed during the early stages of infection and have homologs in other necrotrophic fungal phytopathogens. Functional assays of native recombinant CCL-SSPs indicate that the B. cinerea and S. sclerotiorum single gene homologs are able to induce necrosis when infiltrated into Camellia ‘Nicky Crisp’, Camellia lutchuensis and Nicotiana benthamiana host tissue. In comparison, nine native recombinant CCL-SSP homologs of C. camelliae were unable to replicate the necrosis phenotype when infiltrated into the same host tissue. This work identifies a previously uncharacterized family of fungal necrosis-inducing proteins that appear to have contrasting functions in related host-specific and host-generalist phytopathogens.