A metabolomic approach to dissecting osmotic stress in the wheat pathogen Stagonospora nodorum
Introduction
Fungi typically accumulate polyols in response to osmotic stress. Mannitol, glycerol, erythritol and arabitol have each been implicated in enabling filamentous fungi to withstand osmotic stress (De Vries et al., 2003, Dixon et al., 1999, Fujimura et al., 2000, Liepins et al., 2006, Ruijter et al., 2004, Stoop and Mooibroek, 1998). Arguably, the most comprehensive analysis on the response to osmotic stress in a filamentous fungus has been undertaken on Aspergillus nidulans. A combination of metabolite screening and reverse genetics have demonstrated that glycerol is the primary compatible solute and that a NADP+-dependent glycerol dehydrogenase is required, not glycerol 3-phosphate dehydrogenase (De Vries et al., 2003, Fillinger et al., 2001). Glycerol has also been demonstrated as the primary solute accumulating in response to osmotic stress in Aspergillus oyzae, Neurospora crassa and Hyprocea jecorina.
Plant pathogenic fungi face a hostile growing environment during infection. Plant defence responses, nutrient limitation and oxidative stress are all obstacles that the invading fungus has to overcome. Osmotic stress is also touted as a potential barrier to an invading fungus and consequently the mechanisms enabling tolerance to low water activity are of particular interest. It is often considered that infecting fungal pathogens are likely to undergo osmotic stress during growth in planta. Surprisingly, there are only a handful of reports investigating the metabolic basis of osmotic stress tolerance in fungal plant pathogens. Dixon and co-workers identified that arabitol accumulates during external hyperosmotic stress in the rice pathogen Magnaporthe grisea (Fig. 1) (Dixon, 1999). Metabolite profiling of intracellular metabolites upon exposure of Cladosporium fulvum to salt in vitro revealed the accumulation of arabitol. Subsequent profiling during infection of tomato leaves highlighted an increased abundance of both arabitol and glycerol suggesting that osmoregulation and water acquisition is an important aspect of pathogenicity (Clark et al., 2003).
The ascomycete Stagonospora (syn. Septoria) nodorum (Berk.) Castell. and Germano [teleomorph Phaeosphaeria (syn. Leptosphaeria) nodorum (Müll.) Hedjar.] is a major pathogen on wheat, causing leaf and glume blotch diseases (Solomon et al., 2006a, Weber, 1922). Recent dissection of mannitol metabolism in S. nodorum has shown that the 6-carbon sugar alcohol has no direct role in osmotic stress (Solomon et al., 2007). This raises the question of how does S. nodorum respond to changes in the osmotic state of its growing environment. To address this, S. nodorum was exposed to strong saline conditions to induce osmotic stress and undertook a non-targeted metabolomics approach to understand the metabolic response. Arabitol was identified as the primary compatible solute in S. nodorum. The genetic tractability of S. nodorum was then exploited to dissect 5-carbon polyol metabolism and the role it plays during osmotic stress.
Section snippets
Fungal strains and media
Stagonospora nodorum SN15 was provided by the Department of Agriculture, Western Australia. The fungus was routinely grown on CzV8CS (Czapek Dox agar (Oxoid) 45.4 g l−1, agar 15.0 g l−1, CaCO3 3.0 g l−1, Campbell’s V8 juice 200 ml l−1, casamino acids 100 mg l−1, peptone 100 mg l−1, yeast extract 100 mg l−1, adenine 15 mg l−1, biotin 100 μg l−1, nicotinic acid 100 μg l−1, p-aminobenzoic acid 100 μg l−1, pyridoxine 100 μg l−1, thiamine 100 μg l−1) (Solomon et al., 2006b). Plates were incubated at 22 °C in 12 h cycles of
Metabolite profiling of Stagonospora nodorum undergoing osmotic stress
Stagonospora nodorum was exposed to 0.5 M NaCl for 6 h as described in the Section 2. The intracellular metabolites were extracted and analysed using GC–MS. A GC injection split ratio of 50:1 was used enabling relative quantification of the more abundant metabolites without saturation of the detector or column. Fifty to sixty peaks were typically separated using this high split ratio. Principal component analysis (PCA) was applied to gain insight into the nature of the multivariate metabolite
Discussion
The goal of this study was to characterise the response to osmotic shock in S. nodorum at the metabolite level. This response in turn could then be further scrutinised using reverse genetics to develop strains affected for the predicted metabolite pathways. In some biological systems, physiological discrepancies have been observed when comparing stress responses due to strong saline conditions and other forms of osmotic stress (Miyama and Tada, 2008). We have yet to observe any significant
Acknowledgments
The authors would like to thank the Grains Research and Development Corporation for financial support.
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