Tps1 regulates CCR in response to G6P sensing.
(A) CCR is Tps1 dependent. Strains were grown for 10 days on CM or minimal media supplemented with 10 mM of the appropriate carbon and nitrogen source. Like Δnut1, Δtps1 and Δtps1 Δnut1 strains are unable to utilize nitrate as nitrogen source. However, deleting the TPS1 gene in Δnut1 strains restores growth on proline and glucosamine as nitrogen sources, demonstrating that CCR is inactivated in Δtps1- carrying strains. (B) G6P sensing by Tps1 activates CCR. To mitigate against AA evaporation, best results were obtained when Guy11, Δtps1 and Δtps1::R22G strains were grown for 5 days on 85 mm petri dishes containing either glucose-rich minimal media with 55 mM glucose and 10 mM NH4+ as sole carbon and nitrogen sources, respectively, or the same medium supplemented with 100 mM of the toxic analogue allyl alcohol (AA). Δtps1 strains were sensitive to 100 mM allyl alcohol, indicating they are carbon derepressed (i.e. CCR is inactivated) in the presence of glucose. Like Guy11, Δtps1::R22G strains were not sensitive to 100 mM allyl alcohol, suggesting CCR operates correctly in the Δtps1::R22G G6P sensing strains. (C) G6P sensing by Tps1 is the trigger for CCR resulting in the inhibition of alternative carbon source (Alt C) utilization by M. oryzae.
