Fluorescencia

The establishment of C. camelliae within compatible Camellia ‘Nicky Crisp’ petal tissue resulted in large changes in gene expression (Fig 4.4). This phenotype contrasted with the relatively small gene expression changes observed during the incompatible

Camellia lutchuensis-C. camelliae interaction (Figs. 4.3 & 4.4). Furthermore, the annotated lists of highly expressed genes identified in both interactions were dissimilar from each other (Table 4.4). Taken together, these results highlight the large genotypic differences associated with two ultimately diverse biological processes; host-cell necrosis (compatible) and the host-defense response (incompatible).

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The compatible interaction stimulated the expression of several Camellia defense gene homologs, including a class 1 chitinase, lipoxygenase 5 and a thaumatin (pathogenesis- related protein 5) gene (Fig. 4.3). Plant chitinases are commonly expressed in response to fungal pathogen attack and act to dissolve chitin in the fungal cell wall (Punja & Zhang, 1993). Lipoxygenase 5 catalyzes the formation of oxylipins, which are implicated in defense signaling during fungal pathogen attack (Porta et al., 2002). Thaumatin overexpression in transgenic plants has led to reduced fungal disease incidence (Datta, et al 1999; Mahdavi et al., 2012). Despite the functional significance of these three genes in host-defense, their expression failed to halt C. camelliae

infection. Although it is possible that the timing or amplitude of gene expression of these defense genes was not sufficient to influence infection, it is also plausible that C. camelliae somehow counters the function of these genes through the action of its own fungal effector proteins. Chitin sequestration by fungal chitin binding effectors has previously been shown as a mechanism for suppressing the host-defense response in the apoplastic biotroph, Cladosporium fulvum (van Esse, et al., 2007; Kombrink et al., 2013).

Interestingly, the expression of Camellia defense genes remained comparatively low during the Camellia lutchuensis-C. camelliae interaction. Of particular interest was the increase in gene expression of two Camellia lutchuensis transcription factor homologs,

ODORANTI1 and CBF-like (Table 4.4). ODORANTI1 is involved in the synthesis of benzoids, which are key components of floral scent (Verdonk et al., 2005). It is unknown if ODORANTI1 also functions in plant defense. However, a positive association between scented Camellia species and C. camelliae resistance was observed in this study (Fig. 3.4). CBF-like transcriptions factors are known to be regulated in response to abiotic stresses such as cold, drought, and salinity (Akhatar et al., 2012). In the absence of functional information, the true role of the homologous

ODORANTI1 and CBF-like ESTs expressed in Camellia lutchuensis is yet to be determined.

As alluded to in chapters 1 and 3, two well documented host-responses to fungal pathogen attack are the modification of host cell walls and the induction of ROS

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(Dickman & de Figueiredo, 2013; Bellincampi et al., 2014). Several cell wall and ROS- scavenging gene homologs were highly upregulated during compatible interactions, including two laccases, two pectinesterases, a peroxidase, and an oxidoreductase gene (Table 4.4). Similar ROS-scavenging gene expression patterns might be expected to be observed during the incompatible interaction, as H2O2 accumulation was observed

during histological analyses (Fig. 3.3, Table 3.1). However, the expression of EST homologs of catalase, ascorbic peroxidase and glutathione S-transferase remained relatively unchanged (Fig. 4.4). In addition, ROS-scavenging genes were absent from the list of Camellia lutchuensis genes that were highly upregulated in response to C. camelliae infection (Table 4.4). It is possible that the gene expression of host ROS- scavenging genes is suppressed during incompatible interactions in order to maintain a high-ROS, antimicrobial environment. Plant NADPH oxidases have previously been shown to be active in establishing a ROS environment upon pathogen attack (Dubiella et al., 2013; Torres et al., 2013). Furthermore, the fungal pathogen Magnaporthe oryzae relies upon the action of its own ROS detoxification enzymes to reduce levels of ROS within host tissue, and establish compatibility (Tanabe et al., 2009; Huang et al., 2011). Elucidating the temporal and spatial expression patterns of Camellia lutchuensis

NADPH oxidase homologs would help to elucidate the role of ROS during the incompatible Camellia lutchuensis-C. camelliae interaction.

107 4.4. Conclusions

Sequencing and assembly of the C. camelliae draft genome resulted in 2581 scaffolds. Scaffold 206 had high synteny to the mating type-specific MAT1-1 locus of Botrytis cinerea (strain B05.10), suggesting that C. camelliae may have a heterothallic mating system. Sequencing and assembly of the Camellia lutchuensis mock, Camellia lutchuensis infected, Camellia ‘Nicky Crisp’ mock and Camellia ‘Nicky Crisp’ infected transcriptomes resulted in a large number of putative ESTs, especially in transcriptomes derived from mock-infected tissue. A total of 13245 ESTs were deemed to be specific to C. camelliae based on alignments to the C. camelliae genome and analysis of the differential gene expression data. Several genes were identified in the

Camellia lutchuensis-C. camelliae interaction that may be involved in resistance to C. camelliae, including two putative transcription factors. However, RNA-seq based gene expression data did not include biological replication, due to the high sequencing cost associated with multiple replicates. Therefore, specific changes in gene expression need to be confirmed via additional experimentation. Despite these shortfalls with gene expression interpretation, the transcriptome content was highly informative and can potentially be used for gene prediction and gene discovery investigations. The next chapter of this thesis focuses on utilizing the C. camelliae genome and transcriptome data to predict and characterize the C. camelliae secretome.

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5. Comparative analysis of the secretomes of