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The CCL-SSP family members identified here are cysteine rich, share conserved and unique sequence motifs (Fig. 6.1) and are found within the genomes of fungi that exhibit necrotrophic life stages (Table 6.1). Furthermore, recombinant B. cinerea and S. sclerotiorum CCL-SSP family members are heat stable (Fig. 6.5) and are able to induce host cell necrosis in multiple hosts (Fig. 6.4 & Appendix 9.20). Many of these defining characteristics are shared with select families of previously characterized fungal effector proteins.

The TOX family of fungal effectors from Stagonospora nodorum and Pyrenophora tritici-repentis are generally cysteine rich, are heat stable, are associated with necrotrophic fungi and cause host-cell necrosis within their hosts (Liu et al., 2009; Liu et al., 2012). Sequence conservation varies between each TOX protein, although ToxB has multiple, identical homologs in Pyrenophora tritici-repentis (Martinez et al., 2004). The main difference between the CCL-SSP family and the TOX proteins, is host- specificity. TOX proteins are host-specific toxins (HSTs) (Tan et al., 2010). Their ability to cause necrosis and subsequent infection is dependent on the presence of a host- specific plant gene (Liu et al., 2009; Liu et al., 2012; Tan et al., 2010). Based on the results shown in Figure 6.4 and Appendix 9.20, the B. cinerea and S. sclerotiorum CCL- SSPs are not host-specific toxins as they are able to induce host-cell death in the distinct hosts Camellia ‘Nicky Crisp’, Camellia lutchuensis and Nicotiana benthamiana.

The necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are virulence factors that are conserved in oomycetes, fungi and bacteria (Qutob et al., 2006). Like their name suggests, they function to induce host-cell necrosis and ethylene

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production. They are more common in microorganisms that rely on hemibiotrophic or necrotrophic nutrition and often exist as large gene families, especially in

Phytophthora species (Gijzen & Nurnberger, 2006). A study in Arabidopsis showed that oomycete-derived NLPs were more active in the plant apoplast (Qutob et al., 2006). This observation correlates with their hypothesized function as proteins that interact with the plasma membrane of host cells, resulting in cytolysis (Ottman et al., 2009). NLPs are non-host specific, which is similar to what has been observed for BcSSP2 and SsSSP3. However, there are several distinctive differences between NLPs and the described CCL-SSP family. Only two cysteine residues are conserved in NLPs making the mature protein more heat labile (Qutob et al., 2006). Furthermore, they universally share the sequence motif ‘GHRHDWE’, which has been shown to be involved in necrosis-inducing function (Ottman et al., 2009), and is absent in all of the CCL-SSP homologs. Additional features of NLPs include their light-dependent activity, the restriction of their activity to dicotyledonous plants and their ability to induce ethylene production. These features have not been tested or measured for BcSSP2 or SsSSP3- associated assays. However, many of the species identified as having CCL-SSP homologs are monocot infecting phytopathogens (Table 6.1). The ability of CCL-SSP homologs of monocot-infecting pathogens to induce host-cell necrosis is still to be determined.

The genomes of B. cinerea and S. sclerotiorum each contain two NLP homologs. Mutant studies of the B. cinerea NLP homologs, BcNEP1 and BcNEP2, indicated that they are not required for full virulence (Arenas et al., 2010). S sclerotiorum NLP homologs SsNEP1 and SsNEP2 share the ‘GHRHDWE’ domain with BcNEP1 and BcNEP2

but their role in virulence has not been determined (Dallal et al., 2010). The protein products of all four genes have been shown to induce necrosis in host tissue, much like that observed for BcSSP2 and SsSSP3. However, due to the lack of a ‘GHRHDWE’ domain in BcSSP2 and SsSSP3 these two proteins cannot be classed as NLPs.

The third family of proteins that share similarities to the CCL-SSP family are the homologs of Cladosporium fulvum Ecp2 (Hce2) proteins. Extracellular C. fulvum protein 2 (Ecp2) was originally described in the non-obligate biotroph C. fulvum, as part of a

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group of extracellular proteins that induce an HR in Cf-Ecp resistant tomato lines (Stergiopoulos & de Wit, 2009). Ecp2 has been shown to contribute to the virulence of

C. fulvum, but its fundamental function is still to be resolved (Lauge et al., 1997). The recent finding that Ecp2 has conserved homologs in many other fungi sets it apart from other species-specific fungal effectors of C. fulvum (Stergiopoulos et al., 2012). Hce2 proteins have 4 conserved cysteine residues and conserved exon/intron boundaries. The Mycosphaerella fijiensis Hce2 protein induces necrosis in tomato lines that lack a

C. fulvum resistance gene (Stergiopoulos et al., 2010). Interestingly, the same M. fijiensis Hce2 protein induces an HR in tomato lines that contain a C. fulvum resistance gene. It is hypothesized that the M. fijiensis Hce2 protein induces necrosis and compatibility through its interaction with a host protein. When the host protein is guarded an ETI-associated defense response ensues, resulting in cell death. It was unknown whether M. fijiensis can survive the ETI-associated defense response as assays were performed using an artificial PVX-expression system (Stergiopoulos et al., 2010). It is unclear whether Hce2 homologs in other species share the same function as M. fijiensis Hce2. Hce2 homologs do not share sequence conservation with the CCL- SSP homologs identified here. Furthermore, no Hce2 homologs have been identified in

B. cinerea or S. sclerotiorum, or any other species of the Leotiomycetes (Stergiopoulos et al., 2012).

Generally, members of the TOX, NLP, Ecp2 and CCL-SSP families induce host-necrosis and are confined to fungal species that exhibit necrotrophic life stages. The CCL-SSP family described here shares multiple characteristics with these previously defined fungal effector families. However, differences in sequence information, primary protein structure and host specificity suggest that they are not equivalent.

6.3.3. Ciborinia camelliae CCL-SSP family members are likely to have undergone gene