The carp lncrna is a cars-related regulatory element with broad effects on the fusarium fujikuroi transcriptome

non-coding RNA Article The carP lncRNA Is a carS-Related Regulatory Element with Broad Effects on the Fusarium fujikuroi Transcriptome Javier Pardo-Medina , Gabriel Gutiérrez, M. Carmen Limón and Javier Avalos *   Citation: Pardo-Medina, J.; Gutiérrez, G.; Limón, M.C.; Avalos, J. The carP lncRNA Is a carS-Related Regulatory Element with Broad Effects on the Fusarium fujikuroi Transcriptome. Non-coding RNA 2021, 7, 46. https://doi.org/10.3390/ ncrna7030046 Academic Editors: Simon Conn and Brian D. Gregory Received: 28 April 2021 Accepted: 29 July 2021 Published: 2 August 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Departamento de Genética, Facultad de Biología, Universidad de Sevilla, 41012 Seville, Spain; [email protected] (J.P.-M.); [email protected] (G.G.); [email protected] (M.C.L.) *Correspondence: [email protected]; Tel.: +34-954-557-110 Abstract: Carotenoid biosynthesis in the fungus Fusarium fujikuroi is regulated by environmental factors, with light being the main stimulating signal. The CarS RING-finger protein plays an important role in the downregulation of structural genes of the carotenoid pathway. A recent transcriptomic analysis on the effect of carS mutation identified a gene for a long non-coding RNA (lncRNA) upstream of carS, called carP, the deletion of which results in increased carS mRNA levels and lack of carotenoid production. We have investigated the function of carP by studying the transcriptomic effect of its deletion and the phenotypes resulting from the reintroduction of carP to a deletion strain. The RNA-seq data showed that the loss of carP affected the mRNA levels of hundreds of genes, especially after illumination. Many of these changes appeared to be cascade effects as a result of changes in carS expression, as suggested by the comparison with differentially expressed genes in a carS mutant. Carotenoid production only recovered when carP was integrated upstream of carS, but not at other genomic locations, indicating a cis-acting mechanism on carS. However, some genes hardly affected by CarS were strongly upregulated in the carP mutant, indicating that carP may have other regulatory functions as an independent regulatory element. Keywords: RNA-seq; carP lncRNA; cis-acting mechanism; carotenoid biosynthesis; light regulation 1. Introduction The genus Fusarium encompasses a large group of fungal species widely distributed in all ecosystems, most of them phytopathogens [ 1 , 2 ]. Some of them have been extensively investigated for the damage they cause in agriculture, but they are also compelling research models due to the richness of their secondary metabolism [ 3 ]. A prominent example is the rice pathogen Fusarium fujikuroi, well-known for its ability to produce gibberellins [ 4 ], plant growth-promoting hormones with applications in agriculture. This species can produce many other secondary metabolites, as indicated by the abundance of gene clusters in its genome dedicated to synthesizing a great diversity of compounds [5]. Recently, F. fujikuroi has received special attention as a model for the study of fungal carotenoid synthesis and its regulation [ 6 ]. All the genes involved in the synthesis of its main carotenoid, the carboxylic xanthophyll neurosporaxanthin, have been identified: carB, carRA,carT, and carD. Two of them, carRA and carB, are organized in a cluster (car cluster) together with carX, which encodes an enzyme that cleaves β -carotene to form retinal, and carO, which encodes a rhodopsin that uses retinal as a chromophore. Carotenogenesis in Fusarium is stimulated by light through transcriptional induction of the carRA, carB, and carT genes, and to a lesser extent, carD gene. Carotenoid photoinduction is the best known photoresponse in this fungus, but the details of its molecular mechanism remain largely unknown. The transcriptional induction of car genes by light is mediated by the White-collar protein WcoA. Inactivation of the wcoA gene severely impairs the transcription of these genes and, subsequently, the accumulation of carotenoids. However, carotenoid photoinduction is not completely abolished in the wcoA mutant. The phenotypic characterization Non-coding RNA 2021,7, 46. https://doi.org/10.3390/ncrna7030046 https://www.mdpi.com/journal/ncrna Non-coding RNA 2021,7, 46 2 of 19 of deletion mutants has revealed the accessory participation of two other photoreceptors, the DASH cryptochrome CryD and the small flavoprotein VvdA [ 7 ]. CryD appears to be responsible for a slower response through an unknown post-transcriptional mechanism, while VvdA regulates the activity of WcoA and possibly CryD as well. Fusarium genomes contain genes for other photoreceptors [ 8 ], including two rhodopsins, a phytochrome, and an additional cryptochrome. Except for CarO rhodopsin, involved in spore germination [ 9 ] and pathogenesis [ 10 ], the functions of these photoreceptors in Fusarium remain to be investigated. A distinctive class of F. fujikuroi carotenoid mutants, generically called carS, exhibit intense orange pigmentation under all culture conditions due to the light-independent accumulation of large amounts of neurosporaxanthin and carotenoid precursors [ 11 ]. The carS mutants analyzed are affected in the carS gene (FFUJ_08714), which encodes a protein of the RING-finger family [ 12 ]. Their increase in carotenoid content is caused by the presence of larger amounts of transcripts of the structural genes of the carotenogenic pathway, particularly carRA and carB. The CarS protein is therefore a negative regulator of Fusarium carotenogenesis, although the carS mutation also affects the expression of genes of other cellular processes [ 13 ]. The mechanism of action of CarS is still unknown, but it has sequence similarity to CrgA from Mucor circinelloides. The deletion of crgA results in a carotenoid overproduction phenotype like that of the carS mutants in F. fujikuroi. CgrA exerts its function by modifying target proteins by ubiquitylation [ 14 ], suggesting a similar molecular mechanism for CarS. Contrary to what might be expected from its role as a negative regulator of carotenogenesis, carS mRNA levels increase under illumination, although the induction is less pronounced than that of the structural car genes [ 12 ]. The regulation of carS may be of special relevance in the control of carotenogenesis in F. fujikuroi. 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). Furthermore, an identical phenotype was observed in the close relative F. oxysporum when its equivalent carP sequence was deleted. 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. It is also unknown whether carP is involved only in the control of carotenogenesis or has other regulatory functions. To test this, we have investigated the consequences of carP deletion in the transcriptome of F. fujikuroi. We show that carP affects the expression of many other genes in addition to those of carotenogenesis. Furthermore, we have reintroduced the carP gene into a ∆ carP mutant to check the influence of its genomic location on the restoration of the wild-type phenotype. The results showed that the effect of carP on carotenogenesis is locus dependent and consistent with a cis-acting role on carS regulation. 2. Results 2.1. RNA-seq Analysis of the Effect of ∆carP Mutation To analyze the possible regulatory functions of carP in addition to its presumed effect on the gene carS and the expression of structural carotenoid genes, total RNA was obtained from mycelia of the wild-type strain and the ∆ carP mutant grown in the dark or illuminated for 60 min. This time of exposure to light was previously described as having a great impact on the transcriptome of this fungus [ 13 ]. It should be noted, however, that these conditions are far from the actual conditions in which the fungus thrives in nature, but they have been Non-coding RNA 2021,7, 46 3 of 19 used in previous studies to investigate transcriptional light regulation. The RNA samples were subjected to massive sequencing and the relative amounts of mRNA were determined for all the annotated genes. The sets of transcripts were compared in the different relevant combinations, and the numbers of differentially expressed genes were determined using a threshold of 2x fold change (log2 = 1) (Table 1). Complete lists of genes exhibiting at least a 2x fold change of mRNA levels in all combinations are shown in Supplementary Table S1. The data revealed a total of 1830 light-regulated genes in the wild-type strain, using the aforementioned threshold change in transcript levels between the illuminated and non-illuminated samples. The number of genes influenced by light was appreciably shortened in the ∆ carP mutant, with 3.5-fold and 4.5-fold reductions for upregulated and down-regulated genes, respectively. In other words, about 75% of the transcripts that were upregulated by light in the wild-type strain, and about 81% of those that were downregulated, did not exhibit significant photoregulation in the ∆ carP mutant. Comparison of transcript levels between the ∆ carP mutant and wild-type strain showed a greater number of differentially expressed genes after illumination than in the dark, with a greater abundance of downregulated genes in the mutant. Table 1. Numbers of genes whose expression changed more than two-fold above (upregulated) or below (downregulated) upon comparison of the indicated strains or culture conditions. Upregulated 2%3Downregulated 2%3 Light 1vs. dark in wild-type strain 970 6.4 860 5.7 Light 1vs. dark in ∆carP mutant 270 1.8 192 1.3 ∆carP mutant vs. wild-type strain in the dark 71 0.4 40 0.2 ∆carP mutant vs. wild-type strain after light 1148 1.0 405 2.7 1 60 min illumination. 2 Upregulated (or downregulated) means increased (or decreased) transcript levels in illuminated compared to dark-grown samples, or in the ∆ carP mutant compared to the wild-type strain. 3Percentage referred to the total number (15,097) of genes annotated in the genome. Differences are more clearly seen in graphical representations, such as volcano plots (Figure 1) or scatter plots (Supplementary Figure S1). Even though similar numbers of genes were upregulated and downregulated by light, the greatest changes in mRNA levels were observed mainly among the photoinducible genes. However, the larger effects disappeared in the ∆ carP mutant (compare Figure 1a,b and Supplementary Figure S1a,b), especially for photoinduced genes. On the other hand, consistent with the data displayed in Table 1, comparisons between sets of wild-type strain and ∆ carP mutant transcripts under the same conditions showed a higher impact of the carP mutation after illumination than in the dark (Figure 1c,d and Supplementary Figure S1c,d). Due to the effect of the carP deletion on the regulation of carotenoid synthesis, the points corresponding to the four genes of the car cluster: carRA,carB,caX, and carO, as well as those for the early gene ggs1, the late genes of the synthesis of neurosporaxanthin, carT and carD, and the regulatory gene carS, have been highlighted in Figure 1and Supplementary Figure S1. In accordance with the former results [ 6 ], the ∆ carP mutation not only decreases the response to light of the structural car genes, which is also patent in the comparisons between wild-type strain and ∆ carP mutant under light, but also that of ggs1. 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]. Non-coding RNA 2021,7, 46 4 of 19 Non-coding RNA 2021, 7, 46 4 of 19 mutant both in the dark and after illumination, which confirms our previous observations [15]. Figure 1. Volcano plot representations of global expression data in the indicated pairwise comparisons. (a,b) Effect of 1 h illumination in the wild type (a) and in the ΔcarP mutant (b). DL: downregulated by light; UL: upregulated by light. (c,d) Effect of ΔcarP mutation in the dark (c) or after 1 h illumination (d). DM: downregulated in the ΔcarP mutant; UM: upregulated in the ΔcarP mutant. p-values of 0.05 and 0.01 are indicated with grey horizontal lines. Genes with a p-value < 0.05 are indicated in blue. Fold changes in abscissae are obtained from DESeq2 data. Genes exceeding the log2 values of ±1 are indicated in red. Genes related to carotenoid metabolism are marked with green dots and a number. 1: carX (FFUJ_11801); 2: carRA (FFUJ_11802); 3: carB (FFUJ_11803); 4: carO (FFUJ_11804); 5: carT (FFUJ_07962); 6: carD (FFUJ_07503); 7: ggs1 (FFUJ_07352); 8: carS (FFUJ_08714). 2.2. Genes Affected by Light in the Wild-Type Strain and in the ΔcarP Mutant Comparison of the lists of light-affected genes in the two strains showed that about 90% of the genes photoinduced in the ΔcarP mutant were also photoinduced in the wildtype strain, a proportion that was also very high (ca. 83%) for photorepressed genes (Figure 2). As expected from previous analyses under similar conditions [13], genes induced by light in the wild-type strain were involved in very different physiological processes, including some related to stress. However, the functional categories that were found relevant in genes that exhibited photoinduction in the ΔcarP mutant were enriched for activities related to signaling pathways. Functional assignations were less clear among the pho- Figure 1. Volcano plot representations of global expression data in the indicated pairwise comparisons. ( a , b ) Effect of 1 h illumination in the wild type ( a ) and in the ∆ carP mutant ( b ). DL: downregulated by light; UL: upregulated by light. ( c , d ) Effect of ∆ carP mutation in the dark ( c ) or after 1 h illumination ( d ). DM: downregulated in the ∆ carP mutant; UM: upregulated in the ∆ carP mutant. p-values of 0.05 and 0.01 are indicated with grey horizontal lines. Genes with a p-value < 0.05 are indicated in blue. Fold changes in abscissae are obtained from DESeq2 data. Genes exceeding the log2 values of ± 1 are indicated in red. Genes related to carotenoid metabolism are marked with green dots and a number. 1: carX (FFUJ_11801); 2: carRA (FFUJ_11802); 3: carB (FFUJ_11803); 4: carO (FFUJ_11804); 5: carT (FFUJ_07962); 6: carD (FFUJ_07503); 7: ggs1 (FFUJ_07352); 8: carS (FFUJ_08714). 2.2. Genes Affected by Light in the Wild-Type Strain and in the ∆carP Mutant Comparison of the lists of light-affected genes in the two strains showed that about 90% of the genes photoinduced in the ∆ carP mutant were also photoinduced in the wild-type strain, a proportion that was also very high (ca. 83%) for photorepressed genes (Figure 2). As expected from previous analyses under similar conditions [ 13 ], genes induced by light in the wild-type strain were involved in very different physiological processes, including some related to stress. However, the functional categories that were found relevant in genes that exhibited photoinduction in the ∆ carP mutant were enriched for activities related to signaling pathways. Functional assignations were less clear among the photorepressed genes in the wild-type strain, some of them related to cell differentiation, virulence, and secondary metabolism (Supplementary Figure S2), and no significant functional groups were identified among the photorepressed genes in the ∆carP mutant. Non-coding RNA 2021,7, 46 5 of 19 Non-coding RNA 2021, 7, 46 5 of 19 torepressed genes in the wild-type strain, some of them related to cell differentiation, virulence, and secondary metabolism (Supplementary Figure S2), and no significant functional groups were identified among the photorepressed genes in the ΔcarP mutant. Figure 2. Effect of light in the transcriptomes of the wild type and the ΔcarP mutant. Venn diagrams show the proportion of coincident genes between those upregulated (left) or downregulated (right) by light in both transcriptomes. Funcat categories are shown for the three transcript sets for which significant categories were found. 2.3. Genes Differentially Expressed in the ΔcarP Mutant Compared to the Wild-type Strain As mentioned above, illumination caused an increase in the number of differentially expressed genes in the ΔcarP mutant compared to the wild-type strain. More than half of the genes upregulated or downregulated in the mutant in darkness were also upregulated or downregulated after illumination (Figure 3a). Analysis of the enriched GO categories found those for carotenoid metabolism among the downregulated ones, but only in the dark because of differences in statical relevance due to the large differences in gene numbers (Supplementary Figure S3). Interestingly, a high proportion of the genes downregulated in the ΔcarP mutant under light were upregulated by light in the wild-type strain under the same conditions (Figure 3b), indicating that carP is needed for adequate photoinduction of a large collection of genes. Figure 2. Effect of light in the transcriptomes of the wild type and the ∆ carP mutant. Venn diagrams show the proportion of coincident genes between those upregulated ( left ) or downregulated ( right ) by light in both transcriptomes. Funcat categories are shown for the three transcript sets for which significant categories were found. 2.3. Genes Differentially Expressed in the ∆carP Mutant Compared to the Wild-Type Strain As mentioned above, illumination caused an increase in the number of differentially expressed genes in the ∆ carP mutant compared to the wild-type strain. More than half of the genes upregulated or downregulated in the mutant in darkness were also upregulated or downregulated after illumination (Figure 3a). Analysis of the enriched GO categories found those for carotenoid metabolism among the downregulated ones, but only in the dark because of differences in statical relevance due to the large differences in gene numbers (Supplementary Figure S3). Interestingly, a high proportion of the genes downregulated in the ∆ carP mutant under light were upregulated by light in the wild-type strain under the same conditions (Figure 3b), indicating that carP is needed for adequate photoinduction of a large collection of genes. To obtain information on the possible functions of the genes influenced by carP, we carried out a hierarchical heatmap according to the mRNA levels of the 591 genes that exhibited a change with respect to the wild-type strain in at least one of the two conditions tested (Figure 4). The tree is separated into two main branches, which correspond to genes that were upregulated or downregulated in the ∆ carP mutant relative to the wild-type strain. For a more specific analysis, a discrimination threshold was arbitrarily chosen (red dotted line in the figure) to distinguish five clusters, two corresponding to upregulated genes (I-II) and three to downregulated genes (III-V). The lists of genes in each cluster are described in Supplementary Table S2. Cluster I consists of 145 genes that, in most cases, are downregulated by light in the wild-type strain. Although their expression patterns were quite diverse, they all showed higher transcript levels in the ∆ carP mutant either in the dark or after illumination, and in many cases photorepression was preserved. GO enrichment analysis only found as significant 26 genes belonging to the category “C-compound and carbohydrate metabolism” (Supplementary Table S3). On the other hand, Cluster II includes only 19 genes with a predominance of photoinduction in the wild-type strain, which, like those of cluster I, are Non-coding RNA 2021,7, 46 6 of 19 upregulated in the ∆ carP mutant. Six of the genes were found to be involved in “sugar, glucoside, polyol, and carboxylate anabolism”. Therefore, all genes in clusters I and II with significant associations with GO categories were related to metabolism. Non-coding RNA 2021, 7, 46 6 of 19 Figure 3. Effect of the ΔcarP mutation in the dark or after one hour illumination. (a) Venn diagrams show the proportion of coincident genes between those upregulated (above) or downregulated (below) in the ΔcarP mutant in the dark or after one hour of light. (b) Venn diagram showing the coincidences between genes upregulated by light in the wild-type strain and those downregulated in the ΔcarP mutant. To obtain information on the possible functions of the genes influenced by carP, we carried out a hierarchical heatmap according to the mRNA levels of the 591 genes that exhibited a change with respect to the wild-type strain in at least one of the two conditions tested (Figure 4). The tree is separated into two main branches, which correspond to genes that were upregulated or downregulated in the ΔcarP mutant relative to the wild-type strain. For a more specific analysis, a discrimination threshold was arbitrarily chosen (red dotted line in the figure) to distinguish five clusters, two corresponding to upregulated genes (I-II) and three to downregulated genes (III-V). The lists of genes in each cluster are described in Supplementary Table S2. Figure 4. Hierarchical heatmap for genes influenced by carP in the transcriptomes of the wild type and the ΔcarP mutant. The red line indicates an arbitrary threshold distinguishing five cluster groups, from I to V, indicated on the right. Significant FUNCAT categories found in cluster III are shown on the right. Figure 3. Effect of the ∆ carP mutation in the dark or after one hour illumination. ( a ) Venn diagrams show the proportion of coincident genes between those upregulated (above) or downregulated (below) in the ∆ carP mutant in the dark or after one hour of light. ( b ) Venn diagram showing the coincidences between genes upregulated by light in the wild-type strain and those downregulated in the ∆carP mutant. Non-coding RNA 2021, 7, 46 6 of 19 Figure 3. Effect of the ΔcarP mutation in the dark or after one hour illumination. (a) Venn diagrams show the proportion of coincident genes between those upregulated (above) or downregulated (below) in the ΔcarP mutant in the dark or after one hour of light. (b) Venn diagram showing the coincidences between genes upregulated by light in the wild-type strain and those downregulated in the ΔcarP mutant. To obtain information on the possible functions of the genes influenced by carP, we carried out a hierarchical heatmap according to the mRNA levels of the 591 genes that exhibited a change with respect to the wild-type strain in at least one of the two conditions tested (Figure 4). The tree is separated into two main branches, which correspond to genes that were upregulated or downregulated in the ΔcarP mutant relative to the wild-type strain. For a more specific analysis, a discrimination threshold was arbitrarily chosen (red dotted line in the figure) to distinguish five clusters, two corresponding to upregulated genes (I-II) and three to downregulated genes (III-V). The lists of genes in each cluster are described in Supplementary Table S2. Figure 4. Hierarchical heatmap for genes influenced by carP in the transcriptomes of the wild type and the ΔcarP mutant. The red line indicates an arbitrary threshold distinguishing five cluster groups, from I to V, indicated on the right. Significant FUNCAT categories found in cluster III are shown on the right. Figure 4. Hierarchical heatmap for genes influenced by carP in the transcriptomes of the wild type and the ∆ carP mutant. The red line indicates an arbitrary threshold distinguishing five cluster groups, from I to V, indicated on the right. Significant FUNCAT categories found in cluster III are shown on the right. Cluster III is the major class, covering 357 genes induced by light in the wild-type strain and downregulated in the ∆ carP mutant. This group included genes associated with nine GO categories (Figure 4), corresponding to very diverse functions. The genes for Non-coding RNA 2021,7, 46 7 of 19 carotenoid biosynthesis, which are known to be repressed in the ∆ carP mutant [ 15 ], and other genes regulated by CarS (see Section 2.6), belong to this category. Clusters IV and V include genes that were downregulated in the ∆ carP mutant, but in this case, they were only moderately affected by light (cluster IV) or clearly downregulated (cluster V) in the wild-type strain. No significant GO categories were found in the 49 genes of cluster IV, which included the genes for the nitrate reductase NiaD (FFUJ_12277, repressed about 10-fold in the ∆ carP mutant), and the probable nitrate transport protein CrnA (FFUJ_00934, repressed about 5-fold). On the other hand, the 20 genes of cluster V included 4 genes associated with the GO category “polysaccharide metabolism”. 2.4. Reintegration of carP in a ∆carP Mutant In a recent work, the albino phenotype of the ∆ carP mutant was described [ 15 ]. To confirm that this trait is due to a lack of carP, we reintroduced the wild-type carP gene into a ∆ carP mutant. In F. fujikuroi transformation experiments, plasmids containing homologous sequences are typically integrated into the genome by homologous recombination at the native site or by heterologous recombination at random locations in the genome. Since the mechanism of action of carP is unknown, analysis of both types of transformants could provide valuable information on whether carP RNA acts in cis or in trans. Therefore, we constructed a transformation vector, pRS246carPneo, which contains the native carP sequence (including its presumptive promoter and terminator sequences) and a G418 resistance cassette (neoR). pRS246carPneo was sequenced to ensure the integrity of the carP sequence, and only a change was detected, the loss of a cytidine close to a C-rich segment, at − 166 bp upstream from the carP transcription initiation (marked in Supplementary Figure S4), which was not expected to affect carP function. Protoplasts of the ∆ carP mutant SG268 were incubated with linearized pRS246carPneo plasmid and G418-resistant colonies were selected, resulting in the isolation of 32 presumptive transformants. Ten of them were genetically purified by growth of uninucleate microconidia in three successive steps and analyzed by PCR with different primer combinations to check the presence of the carP gene (Figure 5a and Supplementary Figure S4a). All G418 resistant transformants were confirmed to contain the carP sequence. However, the integration occurred in the native carP locus in only two of them, T3 and T18, as indicated by the expected PCR amplification between the carP and carS sequences from their genomic DNA samples (Figure 5b and Supplementary Figure S4b). This result was confirmed in a Southern-blot hybridization, in which the bands corresponding to the integration of the plasmid by homologous integration upstream of carS were only found in T3 and T18, but not in other representative transformants (Supplementary Figure S4c). The combined data from Southern-blot hybridization and PCR amplification with the appropriate probe and primers strongly support integration of carP upstream to carS at the same distance as in the wild-type strain, in T3, and in T18. However, the Southern blot also showed other hybridizing bands in T3 and T18, indicating additional ectopic plasmid integrations. Transformants T3 and T18, along with T2 and T21, used as controls for lack of carS-linked carP integrations, were used to perform detailed phenotypic analyses. 2.5. Phenotypic Characterization of Transformants Expressing carP in a ∆carP Mutant No differences in growth or morphology were noted between the colonies of the transformants and the parental strain. However, the T3 and T18 strains showed an orange pigmentation, clearly distinguishable from the albino phenotype exhibited by all other transformants, of which T2 and T21 are representative examples (Figure 6a). Carotenoid analyses confirmed the visual phenotype of the mycelium, with increased levels of carotenoids only in T3 and T18 compared to other transformants or the original SG268 ∆ carP mutant (Figure 6b). However, there were differences in the carotenoid content between T3, T18, and the wild-type strain. While the carotenoid production by T3 resembled that of the wild-type strain in the dark, it only accumulated a third of its carotenoid content in the light. In contrast, T18 exhibited a different pattern of carotenoid Non-coding RNA 2021,7, 46 8 of 19 accumulation: its levels were like those of the wild-type strain under light, but were much higher in the dark, similar to those by the same strain under illumination. Regardless of these differences, presumably due to side effects of carP expression at other integration sites or differences in carP transcription in the native locus, it is noteworthy that the ability to produce carotenoids was only recovered when carP was integrated into its own genomic location. The fact that carP must be upstream of carS to exert its regulatory role in carotenogenesis suggests that this lncRNA is a cis-acting regulatory element. Non-coding RNA 2021, 7, 46 8 of 19 showed other hybridizing bands in T3 and T18, indicating additional ectopic plasmid integrations. Transformants T3 and T18, along with T2 and T21, used as controls for lack of carS-linked carP integrations, were used to perform detailed phenotypic analyses. Figure 5. PCR analysis of the reintegration of the carP sequence in SG268. (a) Genomic maps of ectopic and in situ carP integrations in the SG268 ΔcarP mutant. Yellow (hph): hygromycin B resistance cassette that replaced the carP gene in SG268 [15]. (b) Gel electrophoresis of PCR products from candidate transformants to check the insertion of the carP sequence. Wild-type strain DNA (WT) was used as positive control, and SG268 (ΔcarP mutant) or lack of DNA (0) were used as negative controls. PCR amplification products are indicated on the top map with colored arrowheads and dotted lines. The expected PCR sizes are 1245 bp (A, green), and 2194 bp (B, red). 2.5. Phenotypic Characterization of Transformants Expressing carP in a ΔcarP Mutant No differences in growth or morphology were noted between the colonies of the transformants and the parental strain. However, the T3 and T18 strains showed an orange pigmentation, clearly distinguishable from the albino phenotype exhibited by all other transformants, of which T2 and T21 are representative examples (Figure 6a). Carotenoid analyses confirmed the visual phenotype of the mycelium, with increased levels of carotenoids only in T3 and T18 compared to other transformants or the original SG268 ΔcarP mutant (Figure 6b). However, there were differences in the carotenoid content between T3, T18, and the wild-type strain. While the carotenoid production by T3 resembled that of the wild-type strain in the dark, it only accumulated a third of its carotenoid content in the light. In contrast, T18 exhibited a different pattern of carotenoid accumulation: its levels were like those of the wild-type strain under light, but were much higher in the dark, similar to those by the same strain under illumination. Regardless of these differences, presumably due to side effects of carP expression at other integration sites or differences in carP transcription in the native locus, it is noteworthy that the ability to produce carotenoids was only recovered when carP was integrated into its own genomic location. The fact that carP must be upstream of carS to exert its regulatory role in carotenogenesis suggests that this lncRNA is a cis-acting regulatory element. Figure 5. PCR analysis of the reintegration of the carP sequence in SG268. ( a ) Genomic maps of ectopic and in situ carP integrations in the SG268 ∆ carP mutant. Yellow (hph): hygromycin B resistance cassette that replaced the carP gene in SG268 [ 15 ]. ( b ) Gel electrophoresis of PCR products from candidate transformants to check the insertion of the carP sequence. Wild-type strain DNA (WT) was used as positive control, and SG268 ( ∆ carP mutant) or lack of DNA (0) were used as negative controls. PCR amplification products are indicated on the top map with colored arrowheads and dotted lines. The expected PCR sizes are 1245 bp (A, green), and 2194 bp (B, red). The mRNA levels of the structural carB gene, and the regulatory genes, carS and carP, were analyzed in the wild-type strain and the four transformants described above (Figure 6c). The carB mRNA levels matched the carotenoid content of the different strains. Thus, the carB transcript was found in lower amounts in the T2 and T21 transformants than in the wild-type strain, and the levels were like those found in the original SG268 mutant. However, in T3 and T18 the carB mRNA levels were as high after illumination as those of the wild-type strain, and in the case of T18, even higher in the dark, correlating with the high content of carotenoids under these conditions. As expected from the integration of plasmid pRS246carPneo, carP expression was restored in all four transformants, regardless of the genomic location. However, all strains contained a higher content of carP RNA, consistent with the presence of more than one integrated copy, as indicated by the additional bands observed in the Southern blot (Supplementary Figure S4c). As observed in the wild-type strain, carP transcript levels were appreciably higher in the dark than after illumination in all transformants. Non-coding RNA 2021,7, 46 9 of 19 Non-coding RNA 2021, 7, 46 9 of 19 Figure 6. Effects of carP reintegration in the ΔcarP mutant SG268. (a) Aspect of the colonies of the wild-type strain (WT), the ΔcarP mutant SG268, and the transformants T2, T3, T18, and T21, grown for 1 week under light. (b) Carotenoid content in the same strains grown for 1 week in the dark or under light. Differences found to be significant according to the t tests are indicated (p-values, *: p < 0.033; ***: p < 0.001). (c) Transcript levels for the carP, carS, and carB genes in the same strains grown in the dark (blue bars) or exposed to light for 1 h (orange bars). RT-qPCR data show the mean and standard error of three independent experiments. Transcript levels were normalized against those of the β1-tubulin FFUJ_04397 and GPDH FFUJ_13490 genes. Relative mRNA levels are referred to the mRNA content of the wild-type strain in darkness. The mRNA levels of the structural carB gene, and the regulatory genes, carS and carP, were analyzed in the wild-type strain and the four transformants described above (Figure 6c). The carB mRNA levels matched the carotenoid content of the different strains. Thus, the carB transcript was found in lower amounts in the T2 and T21 transformants than in the wild-type strain, and the levels were like those found in the original SG268 mutant. However, in T3 and T18 the carB mRNA levels were as high after illumination as those of the wild-type strain, and in the case of T18, even higher in the dark, correlating with the high content of carotenoids under these conditions. As expected from the integration of plasmid pRS246carPneo, carP expression was restored in all four transformants, regardless of the genomic location. However, all strains contained a higher content of carP RNA, consistent with the presence of more than one integrated copy, as indicated by the additional bands observed in the Southern blot (Supplementary Figure S4c). As observed in the wild-type strain, carP transcript levels were appreciably higher in the dark than after illumination in all transformants. The mRNA levels of carS gene showed a 5- to 10-fold increase after illumination compared to dark controls in all strains tested, a result consistent with former observations for carS regulation [13]. Regardless of the effect of light, the amount of carS mRNA was markedly higher in all transformants, including the carP mutant SG268, compared to those of Figure 6. Effects of carP reintegration in the ∆ carP mutant SG268. ( a ) Aspect of the colonies of the wild-type strain (WT), the ∆ carP mutant SG268, and the transformants T2, T3, T18, and T21, grown for 1 week under light. ( b ) Carotenoid content in the same strains grown for 1 week in the dark or under light. Differences found to be significant according to the ttests are indicated (p-values, *: p< 0.033; ***: p< 0.001). ( c ) Transcript levels for the carP,carS, and carB genes in the same strains grown in the dark (blue bars) or exposed to light for 1 h (orange bars). RT-qPCR data show the mean and standard error of three independent experiments. Transcript levels were normalized against those of the β1 -tubulin FFUJ_04397 and GPDH FFUJ_13490 genes. Relative mRNA levels are referred to the mRNA content of the wild-type strain in darkness. The mRNA levels of carS gene showed a 5- to 10-fold increase after illumination compared to dark controls in all strains tested, a result consistent with former observations for carS regulation [ 13 ]. Regardless of the effect of light, the amount of carS mRNA was markedly higher in all transformants, including the carP mutant SG268, compared to those of the wild-type strain. The lower carS mRNA levels seemed apparent in transformants T3 and T18 compared to T2, T21, and SG268, in the dark, although the amounts were still clearly above those of the wild-type strain. Considering the high carotenoid content of T18, the high mRNA levels of carS in T18 in the dark in relation to the wild-type strain suggest a post-transcriptional regulation that remains to be elucidated. 2.6. Relation of the Effects of carP and carS Mutations In the carP complementation experiments, the carotenoid-producing phenotype was only recovered when the wild-type carP gene was integrated at its own genomic location, suggesting that carP modulates carS expression via a cis-acting mechanism. A recent study of the effect of the carS mutation on the transcriptome showed that CarS influences the expression of a large set of genes [ 13 ]. This hypothesizes that the effects of carP deletion in the transcriptome could be due to its influence on carS expression. To test this hypothesis, Non-coding RNA 2021,7, 46 16 of 19 4.4. Reintegration of Wild-Type carP in SF268 Protoplasts of SG268 were incubated with linear plasmid pRS246-carPneo and 32 transformants were obtained after selection on G418-supplemented medium. After three passes through uninucleate spores, the insertion of the native carP sequence in the candidate transformants was analyzed by PCR (Supplementary Figure S4) using different primer combinations (Table 2). In all transformants, a carP 1.2 kb band was obtained with PS3, indicating the presence of integrated plasmid containing the carP gene. Integration in the native carP locus (in situ integration) was only observed in transformants T3 and T18, as indicated by amplification with PS4 of a 2.2 kb region spanning from the start of carP to an external sequence in the carS promoter region. Southern blot hybridization of genomic DNA from the wild-type strain, SG268 ∆ carP mutant, and four of the transformants (two with in situ integrations of carP and two with ectopic integrations), was carried out using as a probe a DNA segment next to carP (Supplementary Figure S4), included in the complementation plasmid. Genomic DNA samples were digested with restriction enzymes SphI and XhoI. The results in the SphI-digested samples showed a 10.3-kb band in the wild-type strain and a 10.5-kb band in SG268 and the negative transformants, while a 6-kb band was expected in the positive transformants. In samples digested with XhoI, the 4.9-kb band corresponding to the presence of the HygR cassette was found in the SG268 mutant and the transformants with ectopic integration of carP, while the 3.1-kb band expected for the carP sequence was found in the wild-type strain and in the transformants with in situ integration, confirming the results of the PCR tests and the hybridization of the samples digested with SphI (Section 4.5). Thus, transformants T3 and T18 had recovered the deleted carP sequence at its native locus, while T2 and T21 had integrated it in other genomic locations. 4.5. Southern Blot Samples of at least 4 µ g of genomic DNA were digested with restriction enzymes, electrophoresed in a 0.7% agarose gel using a digoxigenin-labelled molecular ladder (DIG marker III o VII, Roche, Germany), and subjected to the Southern protocol using a positively charged nylon membrane (Hybond-N, Amersham, UK). The transfer was performed by capillarity as described [ 27 ]. The probe used in the Southern blot of the F. fujikuroi transformants was obtained by PCR with PS5 (Table 2). Probe was labelled with digoxigenin (PCR DIG Labeling Mix, Merck, Germany) according to manufacturer’s instructions. The membrane was preincubated with DIG Easy Hyb ™ Granules (Roche) buffer solution for 1 h in a glass cylinder in a hybridization oven (HB-100 Hybridizer, UVP, CA, USA). The DIG-labelled probe was then added at 35 ng/mL to fresh buffer solution and incubated overnight. This was followed by two 5-min washes with 2x SSC 0.1% SDS and two 15-min washes with 2x SSC 0.1% SDS. Subsequently, the membrane was equilibrated with maleic buffer and incubated in blocking reagent solution (Roche) for one hour, and a stock of blocking solution with anti-DIG-alkaline phosphatase antibody (1:10,000) (Merck) was added for 30 min. Membrane was washed twice for 15 min with maleic buffer with 0.3% Tween-20, covered with detection buffer, and placed on an acetate. Then, CDP-Star ® ready to use (Roche) was added and the membrane was covered with another acetate. After a short incubation in the dark, the membrane’s signals were detected in the Odyssey Fc Imaging System (LI-COR, Lincoln, NE, USA). 4.6. Expression Studies Total RNA was extracted with Trizol (Invitrogen, Paisley, UK) from 150–200 mg of ground mycelia samples, using the protocol described by the manufacturer. RNA concentrations were quantified with a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). For qPCR measurements, 2.5 µ g RNA sample was treated with DNAse I and retrotranscribed to cDNA with Transcriptor first-strand cDNA synthesis kit (Roche). Final cDNA concentrations were adjusted to 25 ng/ µ L. RT-qPCR analyses were performed in a LightCycler 480 real-time instrument (Roche) using LightCy- Non-coding RNA 2021,7, 46 17 of 19 cler 480 SYBR Green I Master (Roche) following the manufacturer reaction protocol. The genes, primer sets, and amplicon sizes were carB (PS6, 68 bp), carS (PS7, 181 bp), and carP (PS8, 69 bp). Transcript levels of each gene were normalized against those of the β 1-tubulin gene FFUJ_04397 (PS9, 69 bp) and the glyceraldehyde-3-phosphate dehydrogenase (gpdA) gene FFUJ_13490 (PS10, 94 bp). The data were relativized to the value of the wild-type strain grown in the dark, which was taken as 1. 4.7. RNA-seq Methodology The experiments included three independent samples per strain and condition. Twenty µ g of each RNA sample was treated with DNAse I in NucleoSpin RNA kit columns (Macherey-Nagel, Düren, Germany) following manufacturer’s instructions. The absorbance quality parameters (Ratio A260/A280 > 1.8 and A260/A230 > 1.5 and RIN > 8.5) were tested before processing the samples with the Agilent “Chip RNA Plant Nano” protocol by the company Life Sequencing (Valencia, Spain). Samples were sequenced on Illumina’s NextSeq platform in 75 bp single read mode. “Bcl2fastq2” version 2.19.1 provided by Illumina was used for the conversion of “bcl” files into “fastq” sequence files, a program that also removes the sequencing adapters. Quality values, number of readings, and total sequences, are shown in Supplementary Table S4. 4.8. Bioinformatic Analyses Raw reads from all samples were trimmed, filtered, and quality controlled with AfterQC [ 28 ]. The sequences were mapped with Star [ 29 ]. The Integrative Genomics Viewer IGV application (IGV) version 2.8 was used for mapping visualization [ 30 ]. The annotation used was that from the EnsemblFungi database (https://fungi.ensembl.org, accessed on 20 August 2020) for F. fujikuroi IMI58289. The mapped sequences were analyzed using SeqMonk (version 1.45.4, https://github.com/s-andrews/SeqMonk, accessed on 25 July 2021). Quantification was performed using the RNA-seq quantitation pipeline with the improved mRNA annotation generated by Cuffmerge, merging transcripts, and counting reads over exons. Deseq2 tool [ 31 ], implemented in SeqMonk, which needs raw counts for quantitation, was used to compare among conditions. Differentially expressed genes were selected based on criteria combining a log2 fold change of 1 and an FDR-value of 0.05. Log2 RPM (reads per feature per million reads of library) were used for data visualization and intensity tests, which had a minimum FDR-value below 0.05 multiple testing correction applied with a sample size of 100 when constructing the control distributions. Venny 2.1 was used to draw Venn Diagrams (https://bioinfogp.cnb.csic.es/tools/venny/index.html, accessed on 10 April 2021). Gene expression levels were measured as TPM (transcripts per million) to represent the heatmaps. The heatmap figures were performed using the mean between the three samples in TPM for each strain and condition. The data were then log transformed, centered using the mean between them, and hierarchically clustered with Gene Cluster 3.0 [32]. The visualization was performed with Java TreeView3 [33]. FunCat GO enrichment results were obtained from the FungiFun database (https: //elbe.hki-jena.de/fungifun/, accessed on 25 April 2021) using a significance level of 0.05 and testing for enrichment with FDR correction. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ncrna7030046/s1. Figure S1: Scatter plot representations on the effect of light and carP deletion on the Fusarium fujikuroi transcriptome. Figure S2: Funcat categories of the 680 genes downregulated by light in the wild-type strain. Figure S3: Funcat categories of genes differentially expressed in the ∆ carP mutant. Figure S4: Molecular analysis of the reintegration of the carP sequence in SG268 by replacement with a G418 R cassette (neoR gene). Table S1: Data of the genes upregulated or downregulated by light or by ∆ carP mutation. Table S2: Clusters of genes according to their induction or repression pattern in the wild-type strain and ∆ carP mutant. Table S3: Funcat GO categories and genes in each category. Table S4: Data of the samples and sequences used in the RNA-seq analysis. Non-coding RNA 2021,7, 46 18 of 19 Author Contributions: Experimental work, J.P.-M.; bioinformatic analyses, G.G. and J.P.-M.; work supervision, J.A. and M.C.L.; writing—original draft preparation, J.A.; writing—review and editing, J.A. and M.C.L., funding acquisition, J.A. and M.C.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Spanish Government (Project RTI2018-101902-B-I00, granted by FEDER/Ministerio de Ciencia e Innovación–Agencia Estatal de Investigación). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The RNA-Seq data were deposited in NCBI’s Gene Expression Omnibus repository under GEO Series accession number GSE173210. Acknowledgments: J.P.-M. held a fellowship from the Ministerio de Ciencia, Innovación y Universidades (Program “Formación de Profesorado Universitario”). M.C.L. and J.A. are members of the CaRed Excellence Network (BIO2017-90877-REDT) and European COST Action CA15113 EUROCAROTEN. We thank Centro Informático Científico de Andalucía (CICA) for large-scale web hosting and high-performance computing facilities. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Ma, L.J.; Geiser, D.M.; Proctor, R.H.; Rooney, A.P.; O’Donnell, K.; Trail, F.; Gardiner, D.M.; Manners, J.M.; Kazan, K. Fusarium pathogenomics. Annu. Rev. Microbiol. 2013,67, 399–416. [CrossRef] 2. Geiser, D.M.; Aoki, T.; Bacon, C.W.; Baker, S.E.; Bhattacharyya, M.K.; Brandt, M.E.; Brown, D.W.; Burgess, L.W.; Chulze, S.; Coleman, J.J.; et al. One fungus, one name: Defining the genus Fusarium in a scientifically robust way that preserves longstanding use. Phytopathology 2013,103, 400–408. [CrossRef] 3. Desjardins, A.E.; Proctor, R.H. Molecular biology of Fusarium mycotoxins. Int. J. Food. Microbiol. 2007,119, 47–50. [CrossRef] 4. Studt, L.; Tudzynski, B. Gibberellins and the red pigments bikaverin and fusarubin. In Biosynthesis and Molecular Genetics of Fungal Secondary Metabolites; Martín, J.-F., García-Estrada, C., Zeilinger, S., Eds.; Springer: New York, NY, USA, 2014; pp. 209–238. ISBN 978-1-4939-1190-5. 5. Janevska, S.; Tudzynski, B. Secondary metabolism in Fusarium fujikuroi: Strategies to unravel the function of biosynthetic pathways. Appl. Microbiol. Biotechnol. 2018,102, 615–630. [CrossRef] [PubMed] 6. Avalos, J.; Pardo-Medina, J.; Parra-Rivero, O.; Ruger-Herreros, M.; Rodríguez-Ortiz, R.; Hornero-Méndez, D.; Limón, M.C. Carotenoid biosynthesis in Fusarium.J. Fungi 2017,3, 39. [CrossRef] 7. Castrillo, M.; Avalos, J. The flavoproteins CryD and VvdA cooperate with the White Collar protein WcoA in the control of photocarotenogenesis in Fusarium fujikuroi.PLoS ONE 2015,10, e0119785. [CrossRef] 8. Avalos, J.; Estrada, A.F. Regulation by light in Fusarium.Fungal Genet. Biol. 2010,47, 930–938. [CrossRef] [PubMed] 9. García-Martínez, J.; Brunk, M.; Avalos, J.; Terpitz, U. The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination. Sci. Rep. 2015,5, 7798. [CrossRef] 10. Adam, A.; Deimel, S.; Pardo-Medina, J.; García-Martínez, J.; Konte, T.; Limón, M.C.; Avalos, J.; Terpitz, U. Protein activity of the Fusarium fujikuroi rhodopsins CarO and OpsA and their relation to fungus–plant interaction. Int. J. Mol. Sci. 2018 ,19, 215. [CrossRef] [PubMed] 11. Avalos, J.; Cerdá-Olmedo, E. Carotenoid mutants of Gibberella fujikuroi.Curr. Genet. 1987,11, 505–511. [CrossRef] 12. Rodríguez-Ortiz, R.; Limón, M.C.; Avalos, J. Functional analysis of the carS gene of Fusarium fujikuroi.Mol. Genet. Genom. 2013 , 288, 157–173. [CrossRef] [PubMed] 13. Ruger-Herreros, M.; Parra-Rivero, O.; Pardo-Medina, J.; Romero-Campero, F.J.; Limón, M.C.; Avalos, J. Comparative transcriptomic analysis unveils interactions between the regulatory CarS protein and light response in Fusarium.BMC Genom. 2019 ,20, 67. [CrossRef] 14. Silva, F.; Navarro, E.; Peñaranda, A.; Murcia-Flores, L.; Torres-Martínez, S.; Garre, V. A RING-finger protein regulates carotenogenesis via proteolysis-independent ubiquitylation of a White Collar-1-like activator. Mol. Microbiol. 2008 ,70, 1026–1036. [CrossRef] 15. Parra-Rivero, O.; Pardo-Medina, J.; Gutiérrez, G.; Limón, M.C.; Avalos, J. A novel lncRNA as a positive regulator of carotenoid biosynthesis in Fusarium.Sci. Rep. 2020,10, 678. [CrossRef] [PubMed] 16. Díaz-Sánchez, V.; Limón, M.C.; Schaub, P.; Al-Babili, S.; Avalos, J. A RALDH-like enzyme involved in Fusarium verticillioides development. Fungal Genet. Biol. 2016,86, 20–32. [CrossRef] 17. Pardo-Medina, J.; Gutiérrez, G.; Limón, M.C.; Avalos, J. Impact of the White Collar photoreceptor WcoA on the Fusarium fujikuroi transcriptome. Front. Microbiol. 2021,11, 619474. [CrossRef] [PubMed] Non-coding RNA 2021,7, 46 19 of 19 18. Cha, J.; Zhou, M.; Liu, Y. Mechanism of the Neurospora circadian clock, a FREQUENCY-centric view. Biochemistry 2015 ,54, 150–156. [CrossRef] 19. Marente, J.; Avalos, J.; Limón, M.C. 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.Microorganisms 2020,9, 71. [CrossRef] 20. Marente, J.; Ortega, P.; Pardo-Medina, J.; Avalos, J.; Limón, M.C. Modulation of activity of a carotenoid pathway through the use of the TET-on regulatory system: Application in the fungus Fusarium fujikuroi. In Plant and Food Carotenoids: Methods and Protocols; Rodríguez-Concepción, M., Welsch, R., Eds.; Methods in Molecular Biology; Springer: New York, NY, USA, 2020; pp. 343–360. ISBN 978-1-4939-9952-1. 21. Niederer, R.O.; Hass, E.P.; Zappulla, D.C. Long noncoding RNAs in the yeast S. cerevisiae.Adv. Exp. Med. Biol. 2017 ,1008, 119–132. [CrossRef] 22. Martens, J.A.; Laprade, L.; Winston, F. Intergenic transcription is required to repress the Saccharomyces cerevisiae SER3 Gene. Nature 2004,429, 571–574. [CrossRef] 23. Houseley, J.; Rubbi, L.; Grunstein, M.; Tollervey, D.; Vogelauer, M. A ncRNA modulates histone modification and mRNA induction in the yeast GAL gene cluster. Mol. Cell 2008,32, 685–695. [CrossRef] [PubMed] 24. Till, P.; Pucher, M.E.; Mach, R.L.; Mach-Aigner, A.R. A long noncoding RNA promotes cellulase expression in Trichoderma reesei. Biotechnol. Biofuels 2018,11, 78. [CrossRef] 25. Till, P.; Derntl, C.; Kiesenhofer, D.P.; Mach, R.L.; Yaver, D.; Mach-Aigner, A.R. Regulation of gene expression by the action of a fungal lncRNA on a transactivator. RNA Biol. 2020,17, 47–61. [CrossRef] [PubMed] 26. McCluskey, K.; Wiest, A.; Plamann, M. The Fungal Genetics Stock Center: A repository for 50 years of fungal genetics research. J. Biosci. 2010,35, 119–126. [CrossRef] [PubMed] 27. Sambrook, J.; Russell, D.W. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: New York, NY, USA, 2001. 28. Chen, S.; Huang, T.; Zhou, Y.; Han, Y.; Xu, M.; Gu, J. AfterQC: Automatic filtering, trimming, error removing and quality control for Fastq data. BMC Bioinform. 2017,18, 80. [CrossRef] [PubMed] 29. Dobin, A.; Davis, C.A.; Schlesinger, F.; Drenkow, J.; Zaleski, C.; Jha, S.; Batut, P.; Chaisson, M.; Gingeras, T.R. STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 2013,29, 15–21. [CrossRef] 30. Robinson, J.T.; Thorvaldsdóttir, H.; Winckler, W.; Guttman, M.; Lander, E.S.; Getz, G.; Mesirov, J.P. Integrative genomics viewer. Nat. Biotechnol. 2011,29, 24–26. [CrossRef] [PubMed] 31. Love, M.I.; Huber, W.; Anders, S. Moderated estimation of fold change and dispersion for RNA-Seq data with DESeq2. Genome Biol. 2014,15, 550. [CrossRef] 32. de Hoon, M.J.L.; Imoto, S.; Nolan, J.; Miyano, S. Open source clustering software. Bioinformatics 2004,20, 1453–1454. [CrossRef] 33. Saldanha, A.J. Java treeview-extensible visualization of microarray data. Bioinformatics 2004,20, 3246–3248. [CrossRef]