Activity of the CarS RING finger protein and regulatory connections with CwhA and HmbC in Fusarium fujikuroi

Abstract

The genus Fusarium is a heterogeneous group of phytopathogenic fungi that causes diseases in a large diversity of economically important plants. In addition, Fusarium species produce a wide variety of secondary metabolites, several of which have been investigated in Fusarium fujikuroi. Our group investigates in this fungus the molecular mechanisms that regulate the synthesis of carotenoids, a class of lipophilic terpenoid responsible for the yellow, orange, or reddish pigmentation characteristic of many organisms. In F. fujikuroi carotenoid production is up-regulated by light through the photoreceptor WcoA, whereas a putative E3 ubiquitin ligase, the CarS RING Finger protein, is a down-regulator of the biosynthesis. Both regulatory proteins act modulating the mRNA levels of the genes of the carotenoid pathway (car genes). Loss of carS function results in carotenoid overproduction while its overexpression produces an albino phenotype. Previous assays in our group identified potential target proteins for CarS, one of them CwhA, ortholog of Cwh43 of Saccharomyces cerevisiae. Other strategies have been carried out to search for new potential regulators of the car genes, and recently two proteins of the HMG-box family were identified by a biotin-mediated pulldown method as able to bind to car genes promoters. In this Thesis, we investigated the mechanisms of action of the CarS protein and the regulatory connection with the CwhA and HmbC proteins, the latter one of the HMG-box proteins mentioned above. In chapter I, in vitro ubiquitination assays demonstrated the E3 ligase activity of CarS and the capacity of this protein to ubiquitinate purified WcoA in vitro, supporting regulatory connection between CarS and WcoA. Unfortunately, because of its very low amounts, CarS protein was not sufficiently detected in in F. fujikuroi extracts by western blot, which did not encourage to carry immunoprecipitation assays. In Chapter II, the biological role of CwhA was investigated through the study of targeted deletion mutants. The cwhA deletion revealed no significant effects except a lower germination rate of the conidia, suggesting a direct or indirect role of this protein in this process. However, the biological role of CwhA could be determined. The Cwh43 protein of S. cerevisiae is involved in the remodeling of GPI lipids to ceramides, and the expression of cwhA in a cwh43Δ yeast mutant restored this remodeling capacity, indicating the same enzymatic activity for CwhA. The cwhA mutant has minor lipidome alterations, but the mutation did not affect carotenoid biosynthesis in the wild type. However, the mutation doubled the carotenoid accumulation in a carS overproducing mutant, suggesting that the changes in the lipid content affects the ability of the mutant to store the excess of carotenoids. Finally, the function of HmbC protein from the High Mobility Group (HMG) family was also investigated in Chapter III by targeted deletion. The hmbC mutants increased carotenoid production due to higher mRNA levels of the car biosynthetic genes. Furthermore, hmbC deletion resulted in reduced carS mRNA levels, that could also explain the partial deregulation of the carotenoid pathway. Therefore, this protein is involved in the regulation of the carotenoid pathway, directly on the car genes, or indirectly through the control of the expression of gene carS. In both cases these regulatory actions are presumably carried out through an epigenetic mechanism related to chromatin structure, as reported for this class of proteins. The phenotype of the mutants indicates that HmbC participates also in other cellular processes. Thus, developmental alterations under specific stress conditions, or reduced sensitivity to cell wall degrading enzymes indicated by lower protoplast formation, suggest changes in cell wall chemical composition in the hmbC mutants. In summary, this Thesis contributes to elucidate the action mechanisms of the negative regulator of carotenogenesis CarS, and provides information on the function of two other proteins, one identified by its physical interaction with CarS, CwhA, involved in GPI lipids remodeling, and another one that participates in the regulation of carotenoid biosynthesis predictably by an epigenetic regulatory mechanism.