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

1 Scientific RepoRtS | (2020) 10:678 | https://doi.org/10.1038/s41598-020-57529-2 www.nature.com/scientificreports A novel lncRnA as a positive regulator of carotenoid biosynthesis in Fusarium obdulia parra-Rivero1,2, Javier pardo-Medina 1,2, Gabriel Gutiérrez1, M. Carmen Limón1 & Javier Avalos 1* 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 genus Fusarium comprises a large group of phytopathogenic fungi widely distributed in nature. Some species are well-known research models for basic biological processes, such as Fusarium oxysporum in phytopathogenesis1 and Fusarium fujikuroi in secondary metabolism2. F. oxysporum comprises many species-specific plant pathogens, called formae specialis, and some of them have been widely used for the study of pathogenesis mechanisms and fungus-plant interactions3,4. Fusarium fujikuroi was first known for the production of gibberellins, growth-promoting plant hormones, and has later been investigated for the synthesis of other metabolites, such as bikaverin and fusarins5,6. Some Fusarium species are also models for the investigation of carotenoid biosynthesis and its regulation7. Early studies in Fusarium aquaeductuum revealed the accumulation of xanthophyll neurosporaxanthin (NX), a carboxylic apocarotenoid previously discovered in Neurospora crassa. Subsequent studies in Fusarium fujikuroi led to the identification of all the genes of the NX pathway7, carB, carRA, carT, and carD, which respectively encode a desaturase, a bifunctional phytoene synthase/carotene cyclase, a torulene cleaving dioxygenase, and an aldehyde dehydrogenase (Supplementary Fig.S1). The carB and carRA genes are organized in a coregulated cluster with another dioxygenase gene, carX8. The CarX enzyme is capable of cleaving β-carotene, a side product of NX biosynthesis9, into retinal10. A fourth gene in the cluster is carO, which encodes a photoactive rhodopsin that uses retinal as chromophore11. The biosynthesis of carotenoids in Fusarium is upregulated by light12 through transcriptional activation of the structural genes of the pathway, in which the White Collar photoreceptor WcoA plays a central role13. The transcriptional response is severely impaired in the absence of a functional WcoA protein, but there is an apparent accumulation of carotenoids in wcoA mutants under illumination14 that involves the participation of at least a second photoreceptor. This could be DASH cryptochrome CryD13, as indicated by the slower accumulation of carotenoids in ΔcryD mutants despite activation by WcoA13. CryD photoactivity has been demonstrated biochemically15, but only minor effects were found in the mRNA levels of car genes in ΔcryD mutants after illumination, suggesting its participation in a post-transcriptional regulatory mechanism. Another photoreceptor protein 1Department of Genetics, Faculty of Biology, University of Seville, E-41012, Seville, Spain. 2These authors contributed equally: Obdulia Parra-Rivero and Javier Pardo-Medina. *email: [email protected] open 2 Scientific RepoRtS | (2020) 10:678 | https://doi.org/10.1038/s41598-020-57529-2 www.nature.com/scientificreports www.nature.com/scientificreports/ that participates in the regulation of carotenogenesis by light in Fusarium is VvdA, an orthologue of the Vivid protein of N. crassa, which supposedly counteracts WcoA activity16. A class of mutants of F. fujikuroi exhibits deep orange pigmentation in the dark due to the synthesis of large amounts of NX and carotenoid precursors17. These mutants, also found in F. oxysporum and generically called carS, are affected in a single gene that encodes a protein of the RING-finger family18,19. The deep orange phenotype is due to the presence of large amounts of transcripts for the structural genes of the pathway, especially those of the car cluster, which indicates that CarS is a negative regulator of the expression of the car genes. This cluster is not the only regulatory target of CarS; the carS mutation produces noticeable changes in the mRNA levels of hundreds of genes, a notable proportion of them also regulated by light20. The mechanism of action of CarS remains unknown, but RING-finger domains belong to a family of E3-like ligase involved in the labelling of target proteins by ubiquitylation, a regulatory signal that frequently leads to their degradation. The alleged ortholog of CarS in Mucor circinelloides, CrgA, has been the subject of detailed investigation and its ubiquitylation activity has been demonstrated21–23. CarS and CrgA also contain a LON protease domain, whose function has not been determined. Due to its regulatory effects on a large collection of genes, we assume that carS expression may be subject to complex regulation. The carS gene is preceded by a 4-kb upstream sequence without predicted ORFs in genome annotations. A former study of two F. oxysporum T-DNA insertion mutants, which exhibited deregulation of carotenoid biosynthesis, revealed sequence alterations in this 4-kb DNA segment upstream of the carS gene18, and a recent RNA-seq study suggested the occurrence of an unidentified transcript in this genomic region with features consistent with a long noncoding RNA (lncRNA). LncRNAs are polyadenilated RNAs of more than 200 bp in length, transcribed by RNA polymerase II but without protein coding functions. It has been discovered that an increasing number of lncRNAs are involved in important biological processes in higher eukaryotes, such as development or cancer. They modulate the expression of target genes through a variety of mechanisms24–26, associated with their sequences or their structure, involving transcriptional, post-transcriptional, and epigenetic levels. Thus, they can interact with transcription factors, chromatin remodelling factors, microRNAs, or mRNAs, and in some cases they can act as enhancer-like RNAs27. However, there is very little information on the functions of lncRNAs in lower eukaryotes. We have found that the transcript found in the genomic region upstream of carS plays a key positive role in the regulation of carotenoid biosynthesis in F. oxysporum and F. fujikuroi, probably through the control of the carS gene. This is the first functional study of a lncRNA in a species of Fusarium. Results Identification of a transcript upstream of the carS gene. In a search for putative regulatory elements in the 4-kb upstream sequence of carS in F. oxysporum, a bioinformatic microRNA prediction tool identified a putativeprecursor microRNA sequence, which we will call here carP*. This alleged microRNA was not confirmed by a specific RNA-seq study for small RNAs (J. Pardo-Medina, unpublished), but a recent RNA-seq analysis on the effect of light and carS mutations in F. fujikuroi and F. oxysporum20 revealed a transcript in both species, which overlaps with the previous sequence of carP* in F. oxysporum (Fig.1) and which we have called carP. The transcripts of carP have a length of 1195 bp in F. oxysporum and 1357 pb in F. fujikuroi, considering as initial and final nucleotides those present in at least 2 readings of the RNA-seq data in different strains and culture conditions (Supplementary Fig.S2A). To distinguish the versions of carP in both species, we call them Fo-carP and Ff-carP. To determine the orientation of carP transcription, a PCR assay was carried out to identify strand-specific PCR products. For this purpose, we obtained cDNA samples using mixtures of primers for each DNA strand of the carP sequence and we used them as substrates for amplification with specific primers of Fo-carP and Ff-carP. As a control, the same protocol was used to determine the orientation of the β-tubulin gene. The results (Fig.2) indicated that both genes encoding carP are transcribed from the same strand as the neighbouring carS gene. Sequence features of carP. A Clustal alignment between the carP sequences of F. oxysporum and F. fujikuroi revealed high conservation, with 929 matches along an overlapping stretch that covers 1165 bp in Fo-carP and 1238 bp in Ff-carP (Supplementary Fig.S2B), with a non-homogenous distribution of coincidences, and the occurrence of numerous gaps (Fig.3A). To consider their possible coding functions, open reading frames (ORFs) in both chains with a threshold of 25 consecutive amino acids were determined. The results (Supplementary Fig.S3) showed 11 ORFs for the Fo-carP sequence, 7 of them in the forward direction (Fig.3B), and 12 ORFs in Ff-carP, of which 5 in the forward direction (Fig.3C). The longest ORFs encoded 131 residues in Fo-carP and 107 in Ff-carP, located in different regions of carP. ORF F4 of Fo-carP was previously annotated as hypothetical proteins FOTG_04033 in F. oxysporum f.sp. vasinfectum, and FOVG_10518 in F. oxysporum f.sp. pisi, but they were not annotated in other Fusarium genomes. Comparisons of the ORF sequences with those of the protein databases did not identify known conserved sequences or domains. E.g, the best match of the Fo-carP sequence gave an E value of 0.005. Most significantly, with the exception of some short segments (Supplementary Fig.S3), there are no coincident ORFs in the carP sequences between both species. Taking into account the possible occurrence of intron sequences, we achieved BlastX ORF-independent searches with whole carP sequences against the NCBI nr database. Without considering the two annotated carP segments of F. oxysporum, the maximum E value was 0.3, found against a cyclase of Streptomyces sp. As alternative approaches, the coding capacity of carP was verified through other sequence characteristics. Analyses based on 3-base periodicity (Supplementary Fig.S4) or codon usage (Supplementary Fig.S5) did not provide evidence of coding functions. Other methods used to assess the protein-coding potential of carP were two versions of Coding Potential Calculator: CPC28 and CPC229, based on intrinsic sequence features, and RNAcode, based on comparative sequence data30. None of these methods provided any support to the occurrence of putative coding sequences in Fo-carP or Ff-carP (Supplementary TablesS1 and S2). 3 Scientific RepoRtS | (2020) 10:678 | https://doi.org/10.1038/s41598-020-57529-2 www.nature.com/scientificreports www.nature.com/scientificreports/ Taken together, all the data suggest that the carP transcript does not have a protein-coding function and, therefore, could play a role as regulatory RNA. The carP sequence is too long for reliable 2D predictions, but if its function depends on a conserved secondary conformation, the structure could be inferred from comparison of the orthologs of carP in different Fusarium species. We have used the RNAcode procedure, capable of detecting possible coding features based on multiple alignment, to obtain a consensus structure. The alignment was performed with the LocARNA-P method31, using 15 carP sequences (Supplementary Fig.S6 and TableS3), including Fo-carP and Ff-carP. The resulting maximum-likelihood tree (Fig.4) showed that most of the carP sequences investigated are closer to Ff-carP than Fo-carP. The rapid evolution of carP makes it possible to build a reliable phylogenetic tree for such closely related species, which contrasts with the lower resolution of the trees based on gene sequences usually used for this purpose, as those of the Tef1 gene (supplementary Fig.S7). The finding of a consensus secondary structure (Supplementary Fig.S8) is consistent with a hypothetical structural role but does not prove it. Some lncRNAs act directly on mRNAs of the target genes through pairing, affecting positively or negatively their stability or their translation. A search with the Clustal Omega program on possible target mRNAs, such as those of the carS or wcoA/wco1 regulatory genes, or those of the structural genes of carotenogenesis, revealed the absence of sequences of more than 20 bp with more than 50% identity, a threshold below which coincident segments are found in any random gene. This result makes unlikely a direct function of carP by pairing on mRNAs of the genes of interest. occurrence of carP in other species. NCBI megablast with Fo-carP versus the nr database of all species, except fungi, did not give significant matches (E < 10). Similarly, no significant match was obtained with an NCBI megablast with Fo-carP or Ff-carP versus the nr database restricted to all fungi, except those of the genus Fusarium, and the same result was obtained with blastn of Fo-carP in the JGI Mycocosm database. Therefore, carP is specific to Fusarium among the fungi and is not found outside the fungal kingdom. Moreover, the carP sequence is widespread but not universal in Fusarium species: A blastn search with Fo-carP against the 249 Fusarium whole genome sequences (WGS) available at NCBI gave only positive hits in 166, using as selection criteria E < 0.001 and query cover >25% (Supplementary TableS4). Therefore, carP was not identified in 1/3 of the available Fusarium WGS. In contrast, orthologs for the carRA and carB carotenoid pathway genes, as well as for the carS regulatory gene, are found in all of them. However, when carP was present, it exhibited a high sequence conservation. Thus, when compared to at least 97% of the Fo-carP sequence, the percentages of carP identity in other Fusarium species varied from 68,6% in Fusarium verticillioides to 97,1% in Fusarium solani. Genomic organization of carP in relation to carS in Fusarium. The proximity of carP to carS and its transcription on the same strand suggest a functional connection. We revised the information available for Fusarium species on prediction of transcripts in the carS region through the Browser FungiDB, using Fusarium Figure 1. Effect of light (one-hour illumination) and carS mutation on transcript readings according to RNA- Seq data in the genomic region between FOXG_09306 and carS (FOXG_09307) genes in F. oxysporum (panel A) (carS mutant: SX1), and between Ffuj_08715 and carS (FFUJ_08714) genes in F. fujikuroi (panel B) (carS mutant: SG39). The location of a putative microRNA precursor sequencefound in F. oxysporum, called carP*, is indicated with an orange box. In each condition, the profiles obtained with two independent biological samples are shown. The readings were represented with IGV program. 4 Scientific RepoRtS | (2020) 10:678 | https://doi.org/10.1038/s41598-020-57529-2 www.nature.com/scientificreports www.nature.com/scientificreports/ Figure 2. Determination of the orientation of the transcription of carP in F. oxysporum (Fo) and F. fujikuroi (Ff) through the amplification from specific single stranded DNA (ssDNA). SM: size markers. gDNA: amplification of the carP transcript using genomic DNA of the wild strain grown in the dark (positive control) as template; RNA: lack of amplification of the carP transcript using as template total RNA (negative PCR control and DNA contamination control in the ssDNA samples). cDNA (F): lack of amplification of carP from ssDNA obtained from a retrotranscription of total RNA with a mixture of forward carP primers. cDNA (R): amplification of carP from ssDNA obtained from a retrotranscription of total RNA with a mixture of reverse carP primers. Results are also shown for amplification of the β-tubulin gene as a control with a known transcriptional orientation. Lanes cropped from different positions of the original gels are separated by a black line. Full-length blots/gels are presented in Supplementary Fig.S13. The sizes of the expected PCR products are 1099 bp for carP and 1169 bp for β-tubulin gene in F. oxysporum, which correspond to 910 and 693 bp in F. fujikuroi. The primers used are described in Supplementary TableS5A. Genomic DNA and total RNA samples were obtained from wild strains grown for 3 days in darkness. Figure 3. Sequence features of Fo-carP and Ff-carP. (A) Schematic representation of the clustal alignment between Fo-carP (red) and Ff-carP (blue). Gaps resulting from the alignment are indicated. The matching bases are shown below as long grey lines. (B,C) Open reading frames (ORFs) in Fo-carP (B, in red) and Ff-carP (C, in blue). The positions of each of the ORFs are represented in the boxed panels (F: forward, in dark color; R: reverse, in pale color). Residues corresponding to forward ORFs are shown below each boxed panel. The analysis of the ORFs was done through the ORFfinder tool. 5 Scientific RepoRtS | (2020) 10:678 | https://doi.org/10.1038/s41598-020-57529-2 www.nature.com/scientificreports www.nature.com/scientificreports/ oxysporum f. sp. lycopersici 4287 as a reference. The result showed considerable variations in 5′ and 3′-UTR carS extensions between different Fusarium species, as well as carS antisense transcripts from the 3′ extensions of the neighbouring gene in some species (Supplementary Fig.S9). In some cases, the 5′ end of the carS transcript extended to the 4-kb upstream region, and at least in the case of F. verticillioides, it overlapped with the carP sequence. This raises the question of whether Fo-carP and Ff-carP may be the result of eventual transcription of carS from a 5′ distant site, resulting in a long carP-containing 5′-UTR. To test this hypothesis, PCR experiments were carried out with combinations of primers from internal regions of carP and carS to verify their occurrence in single transcripts using cDNA obtained from mycelia of wild type and mutant strains of F. oxysporum and F. fujikuroi, grown in darkness or under light. PCR amplification between the carS and carP sequences was obtained from the genomic DNA, but not from the cDNA of any of the strains and species tested, while the amplifications were detected for the internal carP or carS sequences (Fig.5). We conclude that carP and carS derive from separate transcription events, and that carP is an independent lncRNA. Expression of carP. The RNA-seq data displayed in Fig.1 show that the carS mutation results in enhanced transcript levels for carP in both Fusarium species. However, due to the low content of carP RNA in wild strains, the effect of light was not clear. To learn about the regulation of carP transcription, the effects of light and carS mutation on carP RNA levels were investigated by RT-qPCR in different strains and conditions in F. oxysporum and F. fujikuroi, and the analysis was extended to other relevant genes. In wild F. oxysporum, transcript levels of Fo-carP increased appreciably in light in relation to darkness (Fig.6A). The result was similar with primers for carP* or for another carP sequence, as expected for two regions of the same transcript. Photoinduction was less apparent in the carS mutants SX1 and SX2, which exhibited higher levels of carP RNA in the dark and after illumination. In contrast, exposure to light produced a similar five-fold increase in carS transcript, regardless of the presence of the carS mutation. SX1 and SX2 have point mutations in the coding sequence of carS18, and therefore are expected to contain the complete transcripts of the gene. In the case of SX2, a mutation resulted in a premature stop codon in the carS coding sequence that is expected to generate a truncated polypeptide that lacks approximately 50% of the protein. The results with Fo-carP were similar to those obtained with the structural genes carRA and carB, but in this case the effect of light in the wild strain or the effect of the carS mutation were much greater. As expected, the carotenoid content correlated with the mRNA levels of the carRA and carB genes, but with higher carotenoid content in carS mutants than in the wild strain under illumination. The expression pattern of carP in F. fujikuroi differed from that of F. oxysporum (Fig.6B), a result already suggested by the comparison of their RNA-seq data (Fig.1). Ff-carP RNA levels were hardly affected by light in the wild strain investigated. In this case, to test the effect of the carS mutation, the study focused on a carS mutant, SG39, and its carS-complemented strain SG256, in which a functional carS gene has been reintroduced. carP transcript levels were higher in the carS mutant than in the strains with a functional carS gene, especially without illumination. Figure 4. Maximum-likelihood tree of the carP sequences listed in Fig.S6. The tree derives from multiple alignment based on structures made with the locARNA program used for the RNAcode test. Branch support analysis was evaluated by 1000 ultrafast bootstrap replicates. The tree indicates thatFUS01 is actually a F. oxysporum strain, wrongly classifiedas F. fujikuroi. 6 Scientific RepoRtS | (2020) 10:678 | https://doi.org/10.1038/s41598-020-57529-2 www.nature.com/scientificreports www.nature.com/scientificreports/ phenotype of a ΔcarP and ΔcarP* mutants in F. oxysporum. To determine the function of carP in F. oxysporum, a ΔcarP mutant (SX82) and two ΔcarP* mutants (SX80 and SX81) were obtained. All three mutants had the same morphology and growth rate as the wild strain. However, they exhibited albino mycelia under illumination (SX80 shown as an example in Fig.7A), consistent with a defect in carotenoid production, while presenting a wild-type appearance in the dark. As expected, carotenoid analyses showed a strong photoinduction of the carotenoid content in the wild strain, but only traces of carotenoids in the three mutants in the light, with levels only slightly higher than those in the dark (Fig.7B). To determine whether the phenotype in the light was due to reduced expression of the structural car genes, their mRNA levels were quantified by RT-qPCR in the dark or after one hour of illumination and compared with those of the wild strain. The results showed a sharp decrease in the transcript levels of carRA and carB genes in the ΔcarP mutants compared to the wild strain (Fig.7C). Such descent was similar in the deletion strains ΔcarP and ΔcarP*, indicating that the absence of carP* was sufficient for the total loss of Fo-carP function. However, although the mutants exhibited a 100-fold decrease in the mRNA levels for these genes, a minor photoinduction was still apparent compared to the levels in the dark, which was barely reflected in the actual carotenoid content in the light. On the other hand, the mRNA content of carRA and carB did not suffer significant variations in the dark compared to those of the wild strain, indicating the participation of Fo-carP in the transcriptional photoinduction of the structural car genes in F. oxysporum. The deletion of carP also produced clear reductions in the photoinduction of the carX and carO genes (Fig.7D), organized in a coregulated cluster with carRA and carB (Supplementary Fig.S1). The effect on carO was similar to that of carB, adjacent in the car cluster, and the effect on carX was similar to that of carRA, transcribed divergently from a common regulatory region. In contrast, the effect of carP deletion was hardly apparent for the carT and carD genes (Fig.7E), involved in the last steps of NX production. This is consistent with the lower photoinduction exhibited by these genes compared to carRA and carB, which may explain the poor influence of carP deletion. Interestingly, the transcript levels of the carS gene increased appreciably in the ΔcarP and ΔcarP* mutants, in the dark or after illumination (Fig.7F). However, the ΔcarP* mutation did not affect the levels of the transcription of carP itself. phenotype of ΔcarP mutants in F. fujikuroi. To determine the function of the carP gene in F. fujikuroi, three ΔcarP strains (SG268, SG269, and SG270) were obtained. As already observed in F. oxysporum, the colonies of the three ΔFf-carP strains exhibited morphologies and growth capacities similar to those of the wild strain but lacked the characteristic orange pigmentation in the light (SG268 shown in Fig.8A). However, analysis of their carotenoid contents revealed a more drastic effect on carotenogenesis than that observed in F. oxysporum. The wild strain of F. fujikuroi had detectable amounts of carotenoids in the dark, which increased approximately tenfold under illumination, but the ΔFf-carP mutants contained only traces of carotenoids either in the light or in darkness (Fig.8B). However, mRNA levels for the carRA and carB genes decreased about 100-fold but exhibited Figure 5. (A) PCR verification of carP and carS as independent transcripts in F. oxysporum (left panels) and F. fujikuroi (right panels). Amplifications were performed by PCR on cDNA samples in the indicated strains and conditions using primers from internal sequences of carP (upper panels), internal sequences of carS (lower panels) or connecting internal sequences of carP and carS (central panels). SM: size markers. H2O: test for lack of amplification without template DNA. gDNA: amplification from genomic DNA from the wild strain (positive control), WT: Wild strain. SX1, SX2, and SG39: carS mutants. SG256: SG39 complemented with wild carS. SG268: mutant lacking the carP sequence (ΔcarP) as an additional negative control in F. fujikuroi. Lanes cropped from different positions of the original gels are separated by a black line. Full-length blots/gels are presented in Supplementary Fig.S13. (B) Schematic representation of sizes and locations of PCR products obtained with the indicated primers sets (PS) in F. oxysporum (left) and F. fujikuroi (right). 7 Scientific RepoRtS | (2020) 10:678 | https://doi.org/10.1038/s41598-020-57529-2 www.nature.com/scientificreports www.nature.com/scientificreports/ the same pattern of photoinduction. Interestingly, the ΔFf-carP mutants contained in the light the same amounts of carRA and carB transcripts as the wild strain in the dark, but despite that, their carotenoid content was much lower. In contrast to the effect on carRA and carB, the ΔFf-carP mutants exhibited a modest increase on carS mRNA levels in the dark, which was not apparent after illumination. Figure 6. (A) Effect of light and carS mutation (carS mutants SX1 and SX2) on transcript levels for genes carP, carS, carRA, and carB (light: 1-hour illumination) and carotenoid content (light: continuous illumination) in F. oxysporum. In the case of carP, the RT-qPCR primers were selected from two different carP regions, one of them contained in the carP* sequence. (B) Effect of light (1-hour illumination) or carS mutation (carS mutant SG39 and carS complemented strain SG256) on transcript levels for the carP and carB genes in F. fujikuroi. In both species, for RT-qPCR analyses the strains were grown in DGasn broth for 3 days in the dark (dark bars) and exposed for 1 hour to light (light bars). RT-qPCR data show the mean and standard error of RT-qPCR data from three independent experiments. The relative mRNA levels are referred to the mRNA content of the wild strain in darkness. Differences found to be significant according to the t tests are indicated (P-values, *p < 0.033; **p < 0.002; ***p < 0.001). 8 Scientific RepoRtS | (2020) 10:678 | https://doi.org/10.1038/s41598-020-57529-2 www.nature.com/scientificreports www.nature.com/scientificreports/ Effect of carP deletion on the expression of photoreceptor genes. As already mentioned, the regulation of carotenogenesis by light is mediated in F. fujikuroi by the WcoA photoreceptor, called Wc1 in F. oxysporum32, with the accessory participation of the CryD and VvdA photoreceptors16. For that reason, we investigated whether the reduced photoinduction of car structural genes as a result of the deletion of carP is due to an alteration in expression of the genes for these photoreceptors. The study of mRNA levels of the wcoA/wc1, cryD, and vvdA genes by RT-qPCR in the wild strain and ΔcarP mutants showed the same expression patterns for the three genes in F. fujikuroi and F. oxysporum (Supplementary Fig.S10). The transcript levels of the cryD and vvdA genes were strongly photoinduced, but this photoinduction was maintained in the ΔcarP mutants, indicating that carP is not necessary for this photoresponse. As additional evidence, the effect of carP deletion was also checked in the F. oxysporum FOXG_01269 gene, ortholog of the photoregulated con-10 gene of N. crassa33. FOXG_01269 maintained a robust photoinduction in the ΔcarP mutant, indicating that carP is not a general regulator of photoinducible genes in Fusarium. Discussion Most studies on lncRNAs have been dedicated to higher eukaryotes, especially mammals, but they are present in all taxonomic groups, including plants34 and microorganisms, such as the yeastsSaccharomyces cerevisiae and Schizosaccharomyces pombe35. The occurrence of lncRNAs has also been reported in some filamentous fungi. In N. crassa, a study of massive RNA sequencing revealed 939 lncRNAs, more than half of them antisense of annotated genes36. In Fusarium graminearum, a search identified 2574 lncRNAs, of which 1040 were antisense transcripts, and 547 exhibited differential expression associated to the formation offruiting bodies37. F. oxysporum induces changes in the formation of lncRNAs by plants during the infection process as a defence mechanism38,39, but the presumable changes in lncRNA production in the fungus associated to pathogenesis have not been investigated. To date, only one lncRNA has been characterized in filamentous fungi, HAX1 of T. reesei40, which plays a regulatory role in the expression of cellulases, and possibly other lytic enzymes. The HAX1 gene was identified from phenotypic alterations resulting from insertional mutants, and its function was confirmed by targeted mutation and overexpression, but its mechanism of action remains unknown. In this work, we have demonstrated for the Figure 7. Effect of carP deletion in F. oxysporum. (A) Aspect of surface colonies of the wild strain and a representative ΔcarP mutant grown on minimal medium for 1 week under light. (B) Carotenoid content in the wild strain and mutants ΔcarP* and ΔcarP grown for 1 week in the dark or under light. (C–F) Transcript levels for the carRA, carB, carO, carX, carD, carT, carS, and carP genes in the same strains grown in the dark or exposed for 1 hour to light. RT-qPCR data show the mean and standard error of three independent experiments. Relative mRNA levels refer to the mRNA content of the wild strain in the dark. Differences found to be significant according to the t tests are indicated (P-values, *p < 0.033; **p < 0.002; ***p < 0.001). 9 Scientific RepoRtS | (2020) 10:678 | https://doi.org/10.1038/s41598-020-57529-2 www.nature.com/scientificreports www.nature.com/scientificreports/ first time the participation of a Fusarium lncRNA, that we called carP, in a specific metabolic function. The lack of translation of the ORFs in the lncRNAs is difficult to prove. In our case, the lack of correlation between ORFs in two carP versions investigated: Fo-carP in F. oxysporum and Ff-carP in F. fujikuroi, whose deletions produce similar phenotypes, is a strong indication of the non-participation of a protein in the regulatory function of carP. This lack of correlation is basically due to the occurrence of gains or losses of few bases, as revealed by the alignment of Fo-carP and Ff-carP, which indicates that their sequences have evolved maintaining their function despite the alteration of their ORFs. Accordingly, none of the various informatic tools used to investigate its sequence found any indication of protein coding functions either in Fo-carP or Ff-carP. Considered globally, the available information strongly supports the non-coding nature of carP in Fusarium. The absence of relevant carP matching sequences in predictable target genes suggests that carP does not exert its function by modulating the translation or stability of mRNAs involved in carotenogenesis. Other types of mechanisms, such as those based on a specific three-dimensional conformation of the transcript, are not ruled out. carP RNA could interfere with the function of a regulatory protein, which could be CarS. E.g., carP RNA could bind CarS and block its repressor function, so that the absence of carP would leave CarS free to suppress carotenoid synthesis. In this case, the light could perform its activating function, at least partially, facilitating the binding of carP to CarS. As an example of protein inactivation by binding of a lncRNA, the 600-nt gas5 ncRNA binds to the DNA-binding domain of the glucocorticoid receptor under nutrient starvation and inhibits glucocorticoid-regulated transcription in human cells41. Other mechanisms of action based on carP interaction with regulatory proteins cannot be ruled out. E.g., carP might act as a scaffold to facilitate the assembly of histone modification enzymes and alter the expression of target genes. This is the case of the human HOTAIR lncRNA, which binds to two independent protein complexes and results in histone H3 lysine 27 methylation and lysine 4 demethylation42. A possible regulatory mechanism of lncRNAs is the interference of its transcription on the start of transcription of a neighbor gene from a downstream promoter, resulting in a down-regulation. A well-known example is the regulation of the SER3 gene by the upstream SRG1 ncRNA in S. cerevisiae43. This could be the case of the carP Figure 8. Effect of carP deletion in F. fujikuroi. (A) Aspect of surface colonies of the wild strain and a representative ΔcarP mutant grown on minimal medium for 1 week under light. (B) Carotenoid content in the wild strain and three ΔcarP mutants grown for 1 week in the dark or under light. (C) Transcript levels for the carRA, carB, and carS genes in the same strains grown in the dark or exposed for 1 hour to light. RT-qPCR data show the mean and standard error of three independent experiments. Relative mRNA levels refer to the mRNA content of the wild strain in darkness. Differences found to be significant according to the t tests are indicated (P-values, *p < 0.033; **p < 0.002; ***p < 0.001).