Proteomic assessment of sporulation in the wheat pathogen Stagonospora nodorum

Abstract

Stagonospora nodorum is the causal agent of the wheat disease Stagonospora nodorum blotch (SNB). It is of major economic importance within Australia where, in recent growing seasons, it has caused upwards of A$100 million in losses. The severity of the disease is dependent upon several factors, of which asexual sporulation is a major contributor. The aim of this study was to identify proteins that are differentially abundant between a series of S. nodorum strains across sporulation, and using this knowledge, investigate the role they facilitate within asexual development. A quantitative multiplexed 2D LC-MALDI-TOF/TOF MS analysis of S. nodorum, and three mutants previously identified as perturbed at various stages in the development of asexual sporulation, sch1, stuA, and mpd1, was performed using the iTRAQ 8-plex labelling system. This analysis identified a selection of proteins with possible roles in asexual development. The identified proteins were evaluated against a range of criteria, including postulated roles, gene expression, and across which interactions they were altered. These comparisons resulted in the selection of four proteins for further study. These proteins were an inducible near-UV protein (Uvi1), a formate dehydrogenase (Fod1), a predicted HSCARG dehydrogenase (Hsc1), and lastly, a protein of unknown function, SNOG_08052. The gene encoding for each protein of interest was identified, and through homologous recombination knockout mutants were constructed. Characterisation of the four mutants was performed to determine the role of each of these proteins during asexual sporulation. The first three mutants presented with phenotypes not significantly different from the wildtype strain. The SNOG_08052KO strains showed a reduction in sporulation when grown on MM agar supplemented with sucrose as the sole carbon source. This phenotype was complemented by the addition of mannitol to the media. In addition the SNOG_08052KO strains showed significantly increased enzymatic activities for pathways related to mannitol metabolism and reduced pathogenicity in planta. To investigate the biochemical aspects of this strain further, a metabolite analysis was performed. This identified a number of key differences between the wildtype and knockout strains of S. nodorum. An increase in abundance of the metabolite glucose was accompanied by decreases in the abundances of a range of polyols. This suggests that disruption of SNOG_08052 causes an inability of the mutants to accumulate or process simple sugars. The metabolites mannitol and trehalose, which have previously been linked to sporulation, were differentially abundant within the mutants further confirming their involvement in sporulation. This study has utilised a shotgun proteomics workflow and identified a selection of proteins linked to sporulation, and whilst some of these proteins have previous links to sporulation, many do not. Through the use of reverse genetics this study successfully disrupted the genes encoding for four of those proteins, of which one, SNOG_08052, has been found to play a role in development of asexual sporulation. Metabolic assessment of the non-sporulating SNOG_08052KO mutants revealed the ability to accumulate and utilise specific sugars is central to sporulation.