Regulatory roles of photoreceptors and non-coding RNA in Fusarium fujikuroi

Abstract

The genus Fusarium comprises hundreds of species of pathogenic and saprophytic fungi, usually characterized by a complex secondary metabolism. This includes the production of carotenoids, terpenoid pigments that give a characteristic orange color to their colonies. All the biosynthetic genes have been identified, and two of them are grouped in a coregulated cluster. Light plays a major role in promoting their synthesis by activating their transcription, but their effects on fungal biology are much wider. Light is presumably perceived by F. fujikuroi by a battery of photoreceptors, including members of the White-Collar complex, cryptochromes and photolyases, rhodopsins, and phytochromes. The main photoresponses in fungi are normally dependent on the White-collar complex, and carotenoid biosynthesis in Fusarium mainly depends on it, with WcoA as the light-absorbing component. Another photoreceptor, the DASH cryptochrome CryD, also affects the response of carotenogenesis to light, although the available information suggests its participation in a post-transcriptional regulation. Carotenogenesis is downregulated by a protein of the RING Finger family, called CarS, whose mutation results in a carotenoid overproduction phenotype. The carS gene is preceded by a 4 kb sequence with no known genes, in which the presence of two putative genes for microRNA like precursors had previously been suggested. In this Thesis, a massive sequencing specific for small RNAs has been carried out in F. fujikuroi, which confirmed their occurrence in this fungus. The result was consistent with the existence of a functional RNA interference system, supported by the identification of all the predicted enzymatic components encoded in the genome. This system seems to play a role in the biology of the fungus, as suggests the association between some sRNAs and the sequences of some transposons, indicating their role in silencing of these mobile elements. Deletion of one of the components, the gene for a Dicer protein predictably involved in their generation, showed no phenotype under laboratory conditions, suggesting other roles of the produced sRNAs in processes related with other stages of its life cycle, such as pathogenesis or sexual reproduction. The global sRNA analysis failed to identify possible sRNA elements upstream of carS, contradicting the former hypothesis on the participation of microRNAS in carS regulation. However, a new 1.2 kb transcript, that was denominated carP, was identified in carS upstream region. Its sequence lacks relevant open reading frames and the few that exist do not coincide in the equivalent sequence of the close relative Fusarium oxysporum, indicating that the transcript is a long non-coding RNA (lncRNA). The results showed that carP is independently transcribed from carS, and its deletion produces an albino phenotype due to a drop in transcription of the structural car genes, probably as a result of the higher transcription of carS gene. This phenotype was only complemented by reintegration of the carP gene in the native locus, while its ectopic integration did not allow to recover carotenoid production in the recipient carP mutant, indicating a cis-acting regulatory mechanism for carP on carS expression. Global transcriptomic data showed that carP deletion affects the expression of many genes, most of them predictably through its effect on CarS. However, some of the differentially expressed genes are hardly affected by the carS mutation, pointing to specific regulatory effects of carP on other target genes. Global transcriptomic data after different illumination times revealed a diversity of kinetics patterns in mRNA accumulation in the wild strain. Photoinduced genes exhibited fast, intermediate and late responses, while only intermediate and late responses were found for light-repressed genes. A vast majority of these photoresponses were lost in the wcoA mutant, indicating that WcoA is the main photoreceptor responsible for light regulation in F. fujikuroi. Outstandingly, the wcoA mutation brought about massive changes in the transcriptome, affecting about 20% of the genes. Most of these effects were produced regardless of illumination, indicating that WcoA plays a more general light-independent regulatory role in F. fujikuroi. Outstandingly, many of the genes influenced by WcoA were related to secondary metabolism biosynthetic clusters, raising a biotechnological interest for this protein. Parallel analysis of the effect of light on a cryD deletion mutant revealed less severe transcriptomic effects. However, it resulted in changes in the degrees of photoinduction or photorepression of many genes, suggesting an accessory function of CryD in Fusarium photobiology. The summarized results constitute a significant contribution to the knowledge of the regulation of carotenogenesis in F. fujikuroi and its photobiology and provide further evidence on its molecular complexity.