A novel lncRNA as a positive regulator of carotenoid biosynthesis in Fusarium

Abstract

The fungi Fusarium oxysporum and Fusarium fujikuroi produce carotenoids, lipophilic terpenoid pigments of biotechnological interest, with xanthophyll neurosporaxanthin as the main end product. Their carotenoid biosynthesis is activated by light and negatively regulated by the RING-finger protein CarS. Global transcriptomic analysis identified in both species a putative 1-kb lncRNA that we call carP, referred to as Fo-carP and Ff-carP in each species, upstream to the gene carS and transcribed from the same DNA strand. Fo-carP and Ff-carP are poorly transcribed, but their RNA levels increase in carS mutants. The deletion of Fo-carP or Ff-carP in the respective species results in albino phenotypes, with strong reductions in mRNA levels of structural genes for carotenoid biosynthesis and higher mRNA content of the carS gene, which could explain the low accumulation of carotenoids. Upon alignment, Fo-carP and Ff-carP show 75–80% identity, with short insertions or deletions resulting in a lack of coincident ORFs. Moreover, none of the ORFs found in their sequences have indications of possible coding functions. We conclude that Fo-carP and Ff-carP are regulatory lncRNAs necessary for the active expression of the carotenoid genes in Fusarium through an unknown molecular mechanism, probably related to the control of carS function or expression.

The carP lncRNA Is a carS-Related Regulatory Element with Broad Effects on the Fusarium fujikuroi Transcriptome

“…This gene encodes geranylgeranyl diphosphate synthase, which performs the reaction prior to the synthesis of phytoene, the first carotene in the pathway (see Section 2.6). The carS gene, unlike the other genes already indicated, increases its mRNA levels in the ∆carP mutant both in the dark and after illumination, which confirms our previous observations [15].…”

“…The long upstream sequence of the carS gene has no predicted ORFs in the genome annotation; however, an RNA-seq study revealed an unidentified 1-kb transcript in this genomic region with features consistent with a long non-coding RNA (lncRNA). As implied by the RNA-seq procedure, this RNA, which we call carP, is presumably transcribed by RNA polymerase II and polyadenylated, but lacks protein-coding functions [15]. carP is transcribed from the same strand as the carS gene and is required for efficient expression of the car cluster genes, as indicated by the albino phenotype and the strong reduction in mRNA levels of the structural car genes produced by deletion of carP (∆carP).…”

“…The regulatory function of carP is likely associated with the modulation of carS expression, as indicated by increased levels of carS transcripts in ∆carP mutants [15], which could explain the lower expression of structural car genes. However, the molecular mechanism by which carP performs its function is unknown.…”

Controlled Transcription of Regulator Gene carS by Tet-on or by a Strong Promoter Confirms Its Role as a Repressor of Carotenoid Biosynthesis in Fusarium fujikuroi

“…A partial repression is already operating in the wild strain, with presumable scarce CarS levels, as indicated by the low number of transcripts detected in the RNAseq studies and the high expression of the genes of the carotenoid pathway in the absence of the functional CarS protein. A novel lncRNA gene, located upstream to carS, may be at least partially responsible for the attenuated carS expression [38]. However, mRNA levels do not necessarily correlate with functional protein levels, since regulatory proteins are frequently the subject of interactions with other proteins that generate posttranslational modifications to modulate their activity or trigger their degradation.…”

An Overview on Identification and Regulatory Mechanisms of Long Non-coding RNAs in Fungi

“…For instance, the deletion of the poorly transcribed lncRNA carP ( Fo-carP and Ff-carP in the fungi Fusarium oxysporum and F. fujikuroi , respectively) leads to strong reductions of the carotenoid biosynthesis genes at mRNA levels. However, the content of carS mRNA is maintained at a higher level, indicating that carP is involved in activating the expression of carotenoid genes in Fusarium with an unknown molecular mechanism ( Parra-Rivero et al, 2020 ). Additionally, in the fungal pathogen Cryptococcus neoformans , the lncRNA RZE1 controls the yeast-to-hyphal transition by regulating the key morphogenesis regulator Znf2 ( Chacko et al, 2015 ).…”