A comparative analysis of the heterotrimeric G-protein Gα, Gβ and Gγ subunits in the wheat pathogen Stagonospora nodorum

Abstract

BackgroundIt has been well established that the Gα subunit of the heterotrimeric G-protein in the wheat pathogen Stagonospora nodorum is required for a variety of phenotypes including pathogenicity, melanisation and asexual differentiation. The roles though of the Gγ and Gβ subunits though were unclear. The objective of this study was to identify and understand the role of these subunits and assess their requirement for pathogenicity and development.ResultsG-protein Gγ and Gβ subunits, named Gga1 and Gba1 respectively, were identified in the Stagonospora nodorum genome by comparative analysis with known fungal orthologues. A reverse genetics technique was used to study the role of these and revealed that the mutant strains displayed altered in vitro growth including a differential response to a variety of exogenous carbon sources. Pathogenicity assays showed that Stagonospora nodorum strains lacking Gba1 were essentially non-pathogenic whilst Gga1-impaired strains displayed significantly slower growth in planta. Subsequent sporulation assays showed that like the previously described Gα subunit mutants, both Gba1 and Gga1 were required for asexual sporulation with neither mutant strain being able to differentiate either pycnidia nor pycnidiospores under normal growth conditions. Continued incubation at 4°C was found to complement the mutation in each of the G-protein subunits with nearly wild-type levels of pycnidia recovered.ConclusionThis study provides further evidence on the significance of cAMP-dependent signal transduction for many aspects of fungal development and pathogenicity. The observation that cold temperatures can complement the G-protein sporulation defect now provides an ideal tool by which asexual differentiation can now be dissected.

Dissecting the role of G-protein signalling in primary metabolism in the wheat pathogen Stagonospora nodorum

“…The S. nodorum genome encodes the necessary enzymes for lactose degradation, although the biochemical pathway consists of a number of low specificity enzymes. Importantly, however, as previously demonstrated, S. nodorum can utilize lactose as a sole carbon source (Gummer et al, 2012). The slower growth of the gga1-25 strain on lactose combined with the increased accumulation of this metabolite when grown on glucose suggests that lactose is geared towards anabolism in S. nodorum gga1-25, as may be the case with a number of the gga1 metabolites.…”

“…The recorded metabolite abundances therefore likely reflect both direct and indirect metabolic consequences of the mutation. In a previous study by this laboratory, we demonstrated that ongoing cold stress was sufficient to induce moderate levels of asexual sporulation in the mutant strains (Gummer et al, 2012). As previous sporulation studies in S. nodorum have highlighted the role of primary metabolism in this developmental stage (IpCho et al, 2010;Lowe et al, 2009;Solomon et al, 2005Solomon et al, , 2006cTan et al, 2008), metabolomics was again used to dissect the cold-induced response and identify which metabolites are specifically associated with asexual sporulation.…”

“…At sporulation 4 uC , each of the mutant strains differentiated mature pycnidia and viable asexual spores could be harvested as previously described (Gummer et al, 2012). Metabolites were harvested from all cultures, analysed by GC-MS and the normalized metabolite abundances for each of the strains under all conditions were modelled by PCA (Fig.…”

“…Mutants of S. nodorum harbouring inactive copies of a Ga (Gna1, Ga I class), a Gb (Gba1) or a Gc (Gga1) subunit were only weakly pathogenic at best and displayed a number of other phenotypes in vitro including nitrogen utilization deficiencies, impaired melanization and a decrease in extracellular protease activity (Gummer et al, 2012;Solomon et al, 2004). Another phenotype common to mutants lacking either of the heterotrimeric G-protein subunits listed above was a complete lack of sporulation, either in vitro or in planta.…”

“…Another phenotype common to mutants lacking either of the heterotrimeric G-protein subunits listed above was a complete lack of sporulation, either in vitro or in planta. A recent study by Gummer et al (2012) described how prolonged storage at 4 u C could induce sporulation in all of the mutants. After incubation at 4 u C for 6 weeks, pycnidia appeared containing viable pycnidiospores whilst no pycnidia differentiated after incubation at 22 u C for an equivalent time.…”