Victoriano Garre

Distinct white collar‐1 genes control specific light responses in Mucor circinelloides

Light regulates many developmental and physiological processes in a large number of organisms. The best‐known light response in the fungus Mucor circinelloides is the biosynthesis of β‐carotene. Here, we show that M. circinelloides sporangiophores also respond to light, exhibiting a positive phototropism. Analysis of both responses to different light wavelengths within the visible spectrum demonstrated that phototropism is induced by green and blue light, whereas carotenogenesis is only induced by blue light. The blue regulation of both responses suggests the existence of blue‐light photoreceptors in M. circinelloides. Three white collar‐1 genes (mcwc‐1a, mcwc‐1b and mcwc‐1c) coding for proteins showing similarity with the WC‐1 photoreceptor of Neurospora crassa have been identified. All three contain a LOV (light, oxygen or voltage) domain, similar to that present in fungal and plant blue‐light receptors. When knockout mutants for each mcwc‐1 gene were generated to characterize gene functions, only mcwc‐1c mutants were defective in light induction of carotene biosynthesis, indicating that mcwc‐1c is involved in the light transduction pathway that control carotenogenesis. We have also shown that positive phototropism is controlled by the mcwc‐1a gene. It seems therefore that mcwc‐1a and mcwc‐1c genes control different light transduction pathways, although cross‐talk between both pathways probably exists because mcwc‐1a is involved in the light regulation of mcwc‐1c expression.

Role of the White Collar 1 Photoreceptor in Carotenogenesis, UV Resistance, Hydrophobicity, and Virulence of Fusarium oxysporum

ABSTRACT Knockout mutants of Fusarium oxysporum lacking the putative photoreceptor Wc1 were impaired in aerial hyphae, surface hydrophobicity, light-induced carotenogenesis, photoreactivation after UV treatment, and upregulation of photolyase gene transcription. Infection experiments with tomato plants and immunodepressed mice revealed that Wc1 is dispensable for pathogenicity on plants but required for full virulence on mammals.

A negative regulator of light-inducible carotenogenesis in Mucor circinelloides

Abstract. Mucor circinelloides responds to blue light by activating carotene biosynthesis. Wild-type strains grown in darkness contain minimal amounts of β-carotene because of the low levels of transcription of the structural genes for carotenogenesis. When exposed to a light pulse, the level of transcription of these genes increases strongly, leading to the formation of high concentrations of β-carotene. The crgA gene is involved in the regulation of light-induced carotenoid biosynthesis. This gene, originally identified as a 3′-truncated ORF which causes carotene over-accumulation in the dark, encodes a protein with a cysteine-rich, zinc-binding, RING-finger motif, as found in diverse groups of regulatory proteins. The expression of the crgA gene is activated by a light pulse, with a time course similar to that of the structural genes for carotenogenesis. To understand the regulatory role of the crgA gene in carotenogenesis, we have used a genetic approach based on the construction of crgA null mutants by gene replacement. Lack of the crgA function provokes the over-accumulation of carotenoids both in the dark and the light. Introduction of the wild-type crgA allele into these mutants restores the wild-type phenotype for carotenogenesis. The high levels of carotenoid accumulation shown by the null crgA mutants are correlated with an increase in the expression of carotenogenic structural genes. These results strongly indicate that crgA acts as a negative regulator of light-inducible carotenogenesis in M. circinelloides.

Photobiology in the Zygomycota: multiple photoreceptor genes for complex responses to light.

Light is an environmental signal that modulates many aspects of the biology of zygomycete fungi. Light regulation has been investigated in the zygomycetes Phycomyces blakesleeanus, Mucor circinelloides and Pilobolus crystallinus. Examples of light regulation include the phototropism of the fruiting bodies, the regulation of the development of reproductive structures, and the activation of the biosynthesis of β-carotene. In fungi blue light is perceived by proteins homologous to WC-1, a Neurospora crassa photoreceptor and Zn finger protein that interacts with WC-2 to form a photoresponsive transcription factor complex. Unlike ascomycete and basidiomycete fungi that usually have one wc-1 and one wc-2 gene, several studies have uncovered an unexpected multitude of genes similar to wc-1 and wc-2 in the genomes of several zygomycete fungi. Some of these genes are required for fungal photoresponses, but the function of many of them remains unknown. The presence of multiple wc-1 genes confirms previous suggestions of multiple blue-light photoreceptors in Phycomyces.

Malic enzyme activity is not the only bottleneck for lipid accumulation in the oleaginous fungus Mucor circinelloides

Commercial interest in microbial lipids is increasing due to their potential use as feedstock for biodiesel production. The supply of NADPH generated by malic enzyme (ME; NADP+-dependent; EC 1.1.1.40) has been postulated as being the rate-limiting step for fatty acid biosynthesis in oleaginous fungi, based mainly on data from the zygomycete Mucor circinelloides studies. This fungus contains five genes that code for six different ME isoforms. One of these genes, malA, codes for the isoforms III and IV, which have previously been associated with lipid accumulation. Following a strategy of targeted integration of an engineered malA gene, a stable strain overexpressing malA and showing high ME activity has been obtained, demonstrating the feasibility of this strategy to overexpress genes of biotechnological interest in M. circinelloides. This is the first report showing the integration and overexpression of a gene in Zygomycetes. Unexpectedly, the genetically modified strain showed a lipid content similar to that of a prototrophic non-overexpressing control strain, suggesting that another limiting step in the fatty acid synthesis pathway may have been revealed as a consequence of the elimination of malic enzyme-based bottleneck. Otherwise, the fact that prototrophic strains showed at least a 2.5-fold increase in lipid accumulation in comparison with leucine auxotrophic strains suggests that a wild-type leucine biosynthetic pathway is required for lipid accumulation. Moreover, increasing concentrations of leucine in culture medium increased growth of auxotrophs but failed to increase lipid content, suggesting that the leucine synthesized by the fungus is the only leucine available for lipid biosynthesis. These results support previous data postulating leucine metabolism as one of the pathways involved in the generation of the acetyl-CoA required for fatty acid biosynthesis.

High reliability transformation of the basal fungus Mucor circinelloides by electroporation

Molecular approaches to study the biology of the zygomycete Mucor circinelloides depend mainly on the existence of a polyethylene glycol-based transformation method, which is one of the most efficient in zygomycete fungi. However, the poor reliability and low transformation rates of this method are major obstacles in the molecular study of a number of biological processes. This paper describes an easy and reliable method to transform M. circinelloides protoplasts by electroporation. A high-voltage pulse of 25μF capacitance, 400Ω resistance, and 4kV/cm field strength were seen to be the optimal electrical conditions for delivering DNA into M. circinelloides protoplasts. Under these electrical conditions, successful transformations were carried out with several self-replicative plasmid and strain combinations, producing up to more than 500 transformants per μg DNA. Targeted DNA integration of a transgene (atfA gene of Acinetobacter baylyi) in a particular locus (carRP) was also achieved. This transformation method will considerably facilitate in-depth molecular genetic studies of the biology of this fungus.

A RING‐finger protein regulates carotenogenesis via proteolysis‐independent ubiquitylation of a White Collar‐1‐like activator

Protein ubiquitylation plays a major role in the regulation of cellular processes mainly through proteasome‐dependent degradation, although it has become increasingly clear that it is also involved in other processes. In the fungus Mucor circinelloides, blue light regulates carotene biosynthesis, with this response controlled by crgA and mcwc‐1c genes. CrgA shows characteristics of ubiquitin ligases and represses carotenogenesis in the dark, whereas mcwc‐1c is a white collar 1‐like gene required for its light induction. Another two white collar 1‐like genes have been identified in M. circinelloides: mcwc‐1a, which is involved in phototropism, and mcwc‐1b, of unknown function. Analysis of double knockout mutants generated for crgA and every mcwc‐1 gene demonstrated that crgA and mcwc‐1c regulate carotenogenesis by independent pathways. It was also shown that the effect of crgA on carotenogenesis is mediated by mcwc‐1b, which acts as a carotenogenesis activator. CrgA is involved in proteolysis‐independent mono‐ and di‐ubiquitylation of MCWC‐1b, which results in its inactivation. Regulation of carotenogenesis in M. circinelloides by proteolysis‐independent ubiquitylation suggests that this mechanism of control could be more widespread than previously thought.

Comparison of Biochemical Activities between High and Low Lipid-Producing Strains of Mucor circinelloides: An Explanation for the High Oleaginicity of Strain WJ11

The oleaginous fungus, Mucor circinelloides, is one of few fungi that produce high amounts of γ-linolenic acid (GLA); however, it usually only produces <25% lipid. Nevertheless, a new strain (WJ11) isolated in this laboratory can produce lipid up to 36% (w/w) cell dry weight (CDW). We have investigated the potential mechanism of high lipid accumulation in M. circinelloides WJ11 by comparative biochemical analysis with a low lipid-producing strain, M. circinelloides CBS 277.49, which accumulates less than 15% (w/w) lipid. M. circinelloides WJ11 produced more cell mass than that of strain CBS 277.49, although with slower glucose consumption. In the lipid accumulation phase, activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in strain WJ11 were greater than in CBS 277.49 by 46% and 17%, respectively, and therefore may provide more NADPH for fatty acid biosynthesis. The activities of NAD+:isocitrate dehydrogenase and NADP+:isocitrate dehydrogenase, however, were 43% and 54%, respectively, lower in WJ11 than in CBS 277.49 and may retard the tricarboxylic acid cycle and thereby provide more substrate for ATP:citrate lyase (ACL) to produce acetyl-CoA. Also, the activities of ACL and fatty acid synthase in the high lipid-producing strain, WJ11, were 25% and 56%, respectively, greater than in strain CBS 277.49. These enzymes may therefore cooperatively regulate the fatty acid biosynthesis in these two strains.

The RNAi machinery controls distinct responses to environmental signals in the basal fungus Mucor circinelloides

BackgroundRNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which they derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants.ResultsDeletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway.ConclusionThis work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M. circinelloides during exponential and stationary growth phases and opens up an important avenue for in-depth study of genes involved in the regulation of physiological and developmental processes in this fungal model.

cigA, a light-inducible gene involved in vegetative growth in Mucor circinelloides is regulated by the carotenogenic repressor crgA.

A strategy based on the differential-display technique has been applied to identify and to isolate potential target genes controlled by crgA, a negative regulator of the light-inducible carotenogenesis in the fungus Mucor circinelloides. This approach resulted in the identification and cloning of the gene cigA, whose expression is repressed by the crgA gene. After a light pulse, there is an immediate though transient accumulation of transcripts of cigA gene. A similar result was obtained when mycelia were exposed to continuous illumination, suggesting the existence of a photoadaptation mechanism in M. circinelloides. The protein sequence deduced from the nucleotide sequence of cigA shows homology to the mouse and human GDP-fucose protein O-fucosyltransferases. Null cigA mutants were generated by gene replacement. Lack of cigA function does not affect carotenogenesis but provokes a slight reduction in vegetative growth that is independent of light. Introduction of wild-type cigA alleles into a cigA- strain restored the wild-type phenotype for vegetative growth. These results suggest that cigA is involved in vegetative growth and, moreover, that crgA controls cellular processes other than carotenogenesis.