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

Abstract

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.