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Owing to the immobile nature of plants and their inability to escape pathogens, plants have evolved complex defence mechanisms to protect themselves against threats. Plant defence is often separated into two distinct types. The first recognizes pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors and this activates PAMP-triggered immunity (PTI) and basal defence responses (Jones & Dangl, 2006). Basal defence is a broad range of nonspecific defence responses, which in the majority of cases is effective in preventing further colonization by a wide range of pathogens (Dangl & Jones, 2001). However, certain pathogens have evolved the ability to produce effectors, which overcome PTI, leading to effector-triggered susceptibility (ETS). Here, the second type of plant defence, called effector-triggered immunity (ETI), can be activated. This is initiated inside the cell by the recognition of pathogen effectors by plant resistance-gene products, often nucleotide binding site (NBS)-leucine rich repeat (LRR) proteins (Jones & Dangl, 2006). This defence mechanism involves a hypersensitive response and localized host cell death and is therefore successful against biotrophic pathogens that require living host tissue to survive.
The host mechanisms, though governing the resistance and susceptibility to necrotrophic pathogens, are less well understood. Recently, however, several necrotrophs have been shown to secrete host-specific toxins (HSTs) or effector proteins that play a crucial role in the outcome of disease (Wolpert et al., 2002; Oliver & Solomon, 2010). A single dominant gene often confers host susceptibility to these effectors and the presence of both the susceptibility gene and effector results in ETS and corresponding host cell death, therefore causing disease. Thus it would appear that rather than avoid recognition, necrotrophs willingly exploit host cell death mechanism(s) for their own nutritional gain and subsequent disease success. Hence, interactions between necrotrophic effectors and plant susceptibility genes are termed inverse gene-for-gene interactions (Oliver & Solomon, 2010) and the discovery of ETS in necrotrophic interactions has revealed that these pathogens have evolved more sophisticated mechanisms of causing cell death in their host than originally thought.
The best characterized of these necrotrophic host-specific effector proteins is PtrToxA, originally discovered in Pyrenophora tritici-repentis, the causal agent of tan spot of wheat (Ballance et al., 1989; Tomas et al., 1990; Tuori et al., 1995; Zhang et al., 1997). PtrToxA is a 13.2 kDa protein (Tuori et al., 1995) and has a unique protein sequence harbouring no known functional protein motif (Manning & Ciuffetti, 2005). PtrToxA is internalized in a light-dependent manner into sensitive host mesophyll cells containing Tsn1 (Manning & Ciuffetti, 2005) via an unknown mechanism (Manning et al., 2008). Yeast two-hybrid experiments indicate that the Tsn1 protein does not directly interact with PtrToxA and a receptor has not been identified (Faris et al., 2010). Once internalized, PtrToxA is localized to the chloroplast where it interacts with a chloroplast membrane-associated protein designated ToxA binding protein-1 (ToxABP-1; Manning et al., 2007). Recent microarray studies have established major transcriptional reprogramming in wheat following PtrToxA infiltration (Adhikari et al., 2009; Pandelova et al., 2009). These changes include the induction of plant defence responses, impairment of photosynthetic machinery and increased responses to oxidative stress. However, the role of PtrToxA in inducing these responses is unknown.
Recently, genome sequence analysis of the related wheat pathogen Stagonospora nodorum (Hane et al., 2007) identified a PtrToxA homologue (SnToxA) sharing 99.7% sequence identity, and evidence exists for the lateral transfer of this gene from S. nodorum to P. tritici-repentis some time before 1941 (Friesen et al., 2006). Preliminary studies of SnToxA have indicated that SnToxA and PtrToxA have comparable modes of action and the same gene confers susceptibility (Friesen et al., 2006; Vincent et al., 2011). Although a susceptibility gene, Tsn1 contains features typically associated with classical resistance (R) genes, including NBS and LRR domains (Faris et al., 2010). Two additional dominant susceptibility genes in other species have also been identified to contain these R gene-associated domains; LOV1 in Arabidopsis confers susceptibility to the victorin effector produced by Cochliobolus victoriae (Lorang et al., 2007) and Pc in sorghum confers susceptibility to the PC toxin produced by Periconia circinata (Nagy & Bennetzen, 2008). The cloning and identification of these genes have outlined the similarities between host responses that lead to resistance against biotrophs and those that result in susceptibility to necrotrophs (Lorang et al., 2007; Hammond-Kosack & Rudd, 2008).
A major component of successful plant defence mechanisms is the production of antimicrobial secondary metabolites which are either preformed (phytoanticipans) or pathogen/stress-induced (phytoalexins; Du Fall & Solomon, 2012). It is yet to be established whether these metabolites play a role in SnToxA-induced cell death in wheat. In this study, we undertook comprehensive metabolite profiling of wheat extracts to determine the effect of SnToxA on the secondary metabolism of wheat. LC-MS has been used successfully over recent years to measure hundreds of plant secondary metabolites unable to be detected using more traditional GC-MS methods (von Roepenack-Lahaye et al., 2004; Kanno et al., 2010; Spagou et al., 2010). This technology has enabled a powerful approach to investigate the changes in plant secondary metabolism induced by SnToxA in order to understand the mechanisms behind SnToxA-induced cell death. Plant metabolites altered significantly by SnToxA were functionally characterized in terms of their activity against S. nodorum. This led to the discovery of a number of pathways induced by SnToxA that produce metabolites with novel antifungal activity. These results hold substantial potential for control of S. nodorum by the agricultural community in addition to contributing to our understanding of necrotrophic effectors and ETS.