Figure 9. The Nmr1-3 inhibitor proteins regulate nitrogen and carbon metabolism in response to G6P.

(A) As predicted by our model in Figure 8D, Nitrate reductase activity is dependent on glucose availability. Nitrate reductase activity was determined as described in [21], where strains were grown in CM media for 48 hr before switching to minimal media containing either 55 mM glucose (Glc) or no carbon source (-C), with 10 mM nitrate (NO₃ ⁻) or no nitrogen source (-N). Enzyme activity is given as units of nitrate reductase activity per gram of lyophilized mycelia. Results are the average of at least three independent replicates and bars are standard deviation. (B) NIA1 expression was analyzed in Guy11 and Δnmr1 Δnmr2 Δnmr3 triple mutant strains following growth in CM for 48 hr followed by growth in nitrate minimal media lacking a carbon source for 16 hr. Gene expression results were normalized against expression of the ß-tubulin gene (TUB2). Results are the average of at least three independent replicates, and error bars are the standard deviation. (C) ICL1 and NIA1 gene expression was analyzed in Guy11 and Δnmr1 Δnmr2 Δnmr3 triple mutant strains following growth in CM for 48 hr followed by growth in carbon and nitrogen derepressing minimal media (55 mM xylose+10 mM NO₃ ⁻). Gene expression results were normalized against expression of the ß-tubulin gene (TUB2). Results are the average of at least three independent replicates, and error bars are the standard deviation. (D) To explore how the expression of characterized virulence factors, known to be expressed in nitrogen starvation conditions are controlled, we first confirmed that SPM1 and PTH11 are elevated in expression on 55 mM glucose+10 mM NO₃ ⁻ minimal media compared to growth on 55 mM glucose+10 mM NH₄ ⁺, and that this induction is abolished in Δnut1 strains. Gene expression results were normalized against expression of the ß-tubulin gene (TUB2) and are relative to their expression in NH₄ ⁺-containing minimal media. Results are the average of at least three independent replicates, and error bars are the standard deviation. (E) Having confirmed that SPM1 and PTH11 gene expression is nitrate inducible in a Nut1-dependent manner, we next analyzed whether they were regulated by the Nmr1-3 inhibitor proteins. We looked at the expression of these genes on −C+10 mM NO₃ ⁻ minimal media in Guy11 and the Δnmr1 Δnmr2 Δnmr3 triple mutant strains, and found they were significantly elevated in expression in the latter strain compared to Guy11. Gene expression results were normalized against expression of the ß-tubulin gene (TUB2). Results are the average of at least three independent replicates, and error bars are the standard deviation. (F) Summary of gene regulation discussed in Figure 8 and 9.