These could potentially result from the inefficient use of metabolites or products of metabolism due to blockages or even over-active biochemical pathways. Together with the reduced growth rates on different media, the Gna1, Gba1 and Gga1 mutations appear to have introduced metabolic inefficiencies. In the later observed cultures of S. nodorum gna1, gba1 and gga1, where Proteasome inhibitor pycnidia formation was studied, more intense secretions could be seen. It’s likely that the intensity of media discolouration was heightened by accumulation

over the extended culture period however it may also be that the secretions changed as the cultures’ phenotypes changed. It’s also possible that the increased concentration of secreted metabolites in the culture medium played a role in triggering the formation of pycnidia in these strains. Either

way, the increased presence of secreted metabolites in these strains whilst undergoing pycnidial differentiation adds further interest to the identity of these secreted metabolites. Pathogenicity and asexual sporulation of the S. nodorum gna1, gba1 and gga1 strains The capacity to rapidly increase fungal inoculum density by releasing spores from pycnidia following infection of the wheat plant by S. nodorum is fundamental to the success and consequently the impact of SNB. S. nodorum gna1, gba1 and gga1 were all unable to sporulate during infection of the wheat leaf, however although this defect may slow disease amplification,

sporulation is clearly not a prerequisite for leaf necrosis. The inability for disease caused by infection with the gba1 strain to progress beyond chlorosis ITF2357 cell line however, may implicate necrotrophic effector production in S. nodorum as positively regulated by G-protein signalling through the Gβ subunit Gba1 [14]. It is interesting to note that the requirement of the Gβ and Gγ subunits for infection in different fungal plant pathogens varies. For example, it has been previously demonstrated that GBB1 in Gibberella monoliformis is not required for pathogenicity whist the orthologous protein in the related Fusarium oxysporum is much [19, 20]. Our data clearly show that gene encoding for the Gβ subunit, Gba1, is required for S. nodorum to cause disease on wheat. Whilst sporulation was not observed for the gna1, gba1 or gga1 strains in planta, the observations of asexual sporulation described in vitro are of considerable interest. The capacity for the gna1, gba1 and gga1 strains to develop pycnidia during prolonged incubation at 4°C from an already matured, yet non-sporulating culture adds further interest and VX-689 molecular weight potential for using these strains to dissect these fundamental processes in S. nodorum. The physical characteristics of the mutant pycnidia observed in vitro were also of interest. In S. nodorum SN15, differentiation of cells forming the ostiole of the mature pycnidial wall was observed, but was not seen for the mutant pycnidia.