γ-Glutamyltransferases (GGT) in Colletotrichum graminicola: mRNA and enzyme activity, and evidence that CgGGT1 allows glutathione utilization during nitrogen deficiency.

Gamma-glutamyltransferase (GGT, EC 2.3.2.2) cleaves the γ-glutamyl linkage in glutathione (GSH). Three GGTs in the hemibiotrophic plant pathogen Colletotrichum graminicola were identified in silico. GGT mRNA expression was monitored by quantitative reverse-transcriptase PCR. Expression of all three genes was detected in planta during the biotrophic and necrotrophic stages of infection. Of the three GGTs, CgGGT1 mRNA (from gene GLRG&#95;09590) was the most highly expressed. All three GGT mRNAs were up-regulated in wild type nitrogen-starved germlings in comparison to non-starved germlings. CgGGT1 was insertionally mutagenized in C. graminicola, complemented with the wild type form of the gene, and over-expressed. Enzyme assays of two independent CgGGT1 knockouts and the wild type indicated that CgGGT1 is the major GGT and accounts for 86% and 68% of total GGT activity in conidia and mycelia, respectively. The over-expressing strain had 8-fold and 3-fold more enzyme activity in conidia and mycelia, respectively, than the wild type. In an analysis of the GGT knockout, complemented and over-expressing strains, GGT1 transcript levels are highly correlated (r=0.95) with levels of total GGT enzyme activity. CgGGT1 and CgGGT2 genes in strains that had ectopic copies of CgGGT1 were not up-regulated by nitrogen-starvation, in contrast to the wild type. Deletion or over-expression of CgGGT1 had no effect on mRNA expression of CgGGT2 and CgGGT3. In broth in which 3 and 6mM glutathione (GSH) was the nitrogen source, the CgGGT1 over-expressing strain produced significantly (P<0.0001) more biomass than the wild type and complemented strains, whereas the CgGGT1Δ strains produced significantly (P<0.0001) less biomass than the wild type strain. This suggests that CgGGT1 is involved in utilizing GSH as a nitrogen source. However, deletion and over-expression of CgGGT1 had no effect on either virulence in wounded corn leaf sheaths or GSH levels in conidia and mycelia. Thus, the regulation of GSH concentration is apparently independent of CgGGT1 activity.
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