Análisis funcional de fotoproteínas en Fusarium = Functional analysis of photoproteins in Fusarium

Resumen

The ascomycete Fusarium fujikuroi stands out for its capacity to produce a wide array of secondary metabolites. These compounds exhibit a large chemical diversity and very different biological properties, ranging from pernicious to beneficial for human or animals health, in some cases with biotechnological applications. For this reason, the regulation of secondary metabolism is currently an active area of research. The group of the University of Sevilla where this Thesis has been carried out focuses its attention on the study of the production of F. fujikuroi metabolites, such as carotenoids, gibberellins, bikaverins and fusarins. Their syntheses are controlled by different environmental cues, with light and nitrogen among the most relevant ones. In this Thesis we have analyzed the molecular basis of the regulation by light of carotenoid biosynthesis. Carotenoids are terpenoid pigments that provide colors or exert other biological functions in living beings. As relevant examples, in plants they serve as antenna pigments for photosynthesis, and in animals they are used as signaling and vision molecules. Their role in fungi is under discussion, but in some cases they serve as precursor of other relevant compounds and in others they seem to protect against oxidative stress. Carotenoid photoinduction is a well-characterized response in many fungi, such as Neurospora crassa, Phycomyces blakesleeanus or Mucor circinelloides, where it is performed at transcriptional level of the biosynthetic genes. As found in these fungi, light produces in F. fujikuroi a rapid but transient induction of the expression of the structural genes, in this case carRA, carB, and carT. Former studies with another Fusarium species showed that carotenoid photoinduction is particularly effective with blue light and that the action spectrum of the response suggests the mediation of a photoreceptror with a flavin chromophore. Blue-light responses are coordinated in other fungi by LOV-domain flavin proteins of the WC-1 family. Previous studies in F. fujikuroi revealed that the mutants of the wc-1 orthologous gene wcoA conserve the photoinduction of carotenogenesis, thus involving another photoreceptor(s) in this photoresponse. This Thesis has investigated the biological role of other photoreceptors in the photobiology of this fungus. The genome of F. fujikuroi contains several genes coding for blue light photoreceptors in addition to WcoA. The list includes a small LOV protein (VvdA), a DASH-cryptochrome (CryD), a plant-related cryptochrome (Phr1) and a photolyase (PhlA). This Thesis has been dedicated to the study of the CryD and VvdA photoreceptors in F. fujikuroi, and its possible functional relation with the photoreceptor WcoA, formerly studied by the same group. Regarding the role of CryD, the Thesis shows that the gene is strongly induced by light and that WcoA mediates this induction. Targeted mutation experiments showed that CryD produce similar amounts of carotenoids than the wild type in the light. Unexpectedly, they are affected in the production of other secondary metabolites, such as bikaverins or gibberellins under light and nitrogen starvation. However, no correlation was found with the mRNA levels for the biosynthetic genes, indicating that CryD functions at other regulatory levels. The ?cryD mutants also exhibit developmental alterations, such as production of macroconidia, rarely observed in the wild type strain of F. fujikuroi, and defects in the formation of aerial mycelia. Furthermore we have investigated the biochemical properties of CryD, and the results revealed that CryD binds the expected chromophore molecules, FAD and MTHF. Moreover it was found to be a functional photoprotein able to bind nucleic acids and to photorepair UV-induced DNA damages in vitro. In N. crassa, carotenoid biosynthesis is negatively regulated by VVD, a photoreceptor that counteracts the activity of the light-induced White Collar complex, a process known as photoadaptation. Consequently, the mutants of the vvd gene of this fungus produce more carotenoid under light than the wild type. As found for cryD, the expression of the vvd orthologous gene of F. fujikuroi, vvdA, is induced by light through the activity of the WcoA photoreceptor. In contrast to the N. crassa vvd mutants, the ?vvdA mutants of F. fujikuroi accumulate less carotenoids than the wild type, but the transcription pattern of the carotenoid genes is not affected. These results indicate that in F. fujikuroi VvdA does not mediate the photoadapation of carotenogenesis. However our phenotypic studies revealed that VvdA is also involved in development, with noticeable effects of its mutation on conidiation and mycelia organization. Finally, this Thesis has studied the roles of the investigated photoreceptors in the induction kinetics of carotenoid biosynthesis upon illumination of dark-grown mycelia. We found that the induction of carotenogenesis of wild type F. fujikuroi is achieved in two steps, a fast one lasting up to 6 hours, and a slower second one starting after 12 hours. The mutant phenotypes show that the rapid initial activation (1st step) is transcriptionally achieved by the WcoA photoreceptor, which exhibits a high light sensitivity, while the slower delayed activation (2nd step), possibly pot-transcriptional, is mediated by the CryD photoreceptor, which exhibits a low light sensitivity. Both steps are separated by a pause of several hours. Finally, VvdA plays a modulating role, achieving a negative regulation on WcoA in the first stage and a positive regulation on CryD in the second stage. Further analyses of WcoA revealed that it regulates the synthesis of carotenoids in the dark, as already found for bikaverin biosynthesis, a function achieved at transcription level. In summary, we have determined that the F. fujikuroi photoreceptors CryD and VvdA play coordinated roles with WcoA in the photoregulation of carotenoid biosynthesis, and additionally they play light-dependent regulatory functions in different developmental processes. On the other hand, WcoA plays a central role in the regulation of carotenoid biosynthesis, since it is required for the expression of the structural genes in the dark, and for their photoinduction in the first hours of light; additionally WcoA controls the expression of the photoreceptor genes cryD and vvdA.