In this study we have isolated genes that encode

In this study, we have isolated 15 that encode ACL from G. zeae through random mutagenesis by restriction enzyme-mediated integration (REMI). The mutant Z39P186 where the ACL gene had been disrupted showed defects in vegetative growth, asexual and sexual development, virulence, and trichothecene production. We hypothesized that the pleiotrophic changes observed in this mutant resulted from a defect in lipid biosynthesis, since acetyl-CoA is a primary precursor of lipids and ACL is a key enzyme in the generation of acetyl-CoA. The objectives of this study were to determine whether ACL is required for de novo lipid biosynthesis and to elucidate how ACL controls development of G. zeae. The results of this study demonstrate that ACL genes play pivotal roles in fungal development and have increased our understanding of sexual development in G. zeae.
Materials and methods
Results
Discussion Several fungi in the Pezizomycotina subphylum have two genes that encode a mammalian ACL homologue, ACLY, but most species belonging to the Saccharomycotina subphylum do not have ACL genes. In this study we found two ACL genes in G. zeae, ACL1 and ACL2 that matched the C- and N-terminal sequences of ACLY, respectively. G. zeae mutants in either of these genes did not produce any initial structures for fruiting bodies and showed severe reduction in vegetative growth and conidiation. Our results are consistent with the phenotypes found in ACL mutants of Aspergillus nidulans (Hynes and Murray, 2010) but differ from those of S. macrospora (Nowrousian et al., 1999, Nowrousian et al., 2000). Mutations in ACL genes of S. macrospora did not affect vegetative growth and the mutants still produced initial structure of perithecia even though they completely lost fertility (Nowrousian et al., 1999, Nowrousian et al., 2000). The differences may be caused by a difference in the level of cytosolic acetyl-CoA. ACL is a key enzyme in the production of cytosolic acetyl-CoA but there are alternative pathways to produce it (Lehninger et al., 1993). ACS mediates the synthesis of acetyl-CoA from acetate, and our results showed that transcriptions of those genes are independent of ACL (Fig. 9). Exogenous treatment of acetate fully restored vegetative growth and conidiation but not in sexual reproduction and virulence. These results suggest that the deficiency of cytosolic acetyl-CoA in ACL-deleted mutants of G. zeae could be partially complemented by an alternative pathway and that G. zeae is more dependent on ACL-mediated acetyl-CoA production than S. macrospora. In this study, we hypothesized that ACL is a key enzyme in the biosynthesis of triglycerides that accumulate in fungal hyphae during perithecia development and that the loss of fertility observed in ACL-deleted mutants is caused by a deficiency of triglycerides. Genes including ACL involved in lipid biosynthesis were highly expressed during growth and early sexual development in cultures, and during colonization in wheat stalks (Guenther et al., 2009). However, we found that ACL is not required for triglyceride synthesis. The accumulation of lipids in ACL deletion mutants suggests that G. zeae possesses salvage pathways that are able to synthesize acetyl-CoA. Saccharomyces cerevisiae does not have genes encoding ACL, however it does produce cytosolic acetyl-CoA through two ACSs that are localized in the mitochondria, cytosol, and peroxisomes (Takahashi et al., 2006). G. zeae has three homologs of acetyl-CoA synthetases (ACS1–ACS3), and all of them were expressed during the vegetative growth stage when active lipid biosynthesis occurs. The translocation of acetyl-CoA from peroxisomes or mitochondria to the cytosol through carnithine acetyl transferases (CATs) could be an alternative pathway for cytosolic acetyl-CoA. CAT activities have been detected in A. nidulans and both ACL and CAT were reciprocally regulated by carbon sources (Adams et al., 2002, Wynn et al., 1998). G. zeae has two genes that encode CAT, and the expression profile of these genes was similar to that of ACS1–ACS3. Therefore, ACSs and CATs may be the major pathways of acetyl-CoA generation for fatty acid biosynthesis in G. zeae.