The zinc cluster transcription factor Rha1 is a positive filamentation regulator in Candida albicans

Zinc cluster transcription factors are essential fungal regulators of gene expression. In the pathogen Candida albicans, the gene orf19.1604 encodes a zinc cluster transcription factor regulating filament development. Hyperactivation of orf19.1604, which we have named RHA1 for Regulator of Hyphal Activity, generates wrinkled colony morphology under non-hyphal growth conditions, triggers filament formation, invasiveness, and enhanced biofilm formation and causes reduced virulence in the mouse model of systemic infection. The strain expressing activated Rha1 shows up-regulation of genes required for filamentation and cell-wall-adhesion-related proteins. Increased expression is also seen for the hyphal-inducing transcription factors Brg1 and Ume6, while the hyphal repressor Nrg1 is downregulated. Inactivation of RHA1 reduces filamentation under a variety of filament-inducing conditions. In contrast to the partial effect of either single mutant, the double rha1 ume6 mutant strain is highly defective in both serum- and Spider-medium-stimulated hyphal development. While the loss of Brg1 function blocks serum-stimulated hyphal development, this block can be significantly bypassed by Rha1 hyperactivity, and the combination of Rha1 hyperactivity and serum addition can generate significant polarization even in brg1 ume6 double mutants. Thus, in response to external signals, Rha1 functions with other morphogenesis regulators including Brg1 and Ume6, to mediate filamentation.

Modular and Molecular Optimization of a LOV (Light–Oxygen–Voltage)-Based Optogenetic Switch in Yeast

Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light–oxygen–voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call “HAP-LOV”, displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.

Azole resistance is mediated by integration of sterol gene regulation and membrane transporter production by the zinc cluster-containing transcription factor Upc2A in Candida glabrata

The most commonly used antifungal drugs are the azole compounds that interfere with biosynthesis of the fungal-specific sterol: ergosterol. The pathogenic yeast Candida glabrata commonly acquires resistance to azole drugs like fluconazole via mutations in a gene encoding a transcription factor called PDR1 . These PDR1 mutations lead to overproduction of drug transporter proteins like the ATP-binding cassette transporter Cdr1. In other Candida species, mutant forms of a transcription factor called Upc2 are associated with azole resistance, owing to the important role of this protein in control of expression of genes encoding enzymes involved in the ergosterol biosynthetic pathway. Recently, the C. glabrata Upc2A factor was demonstrated to be required for normal azole resistance, even in the presence of a hyperactive mutant form of PDR1 . Using genome-scale approaches, we define the network of genes bound and regulated by Upc2A. By analogy to a previously described hyperactive UPC2 mutation found in Saccharomyces cerevisiae , we generated a similar form of Upc2A in C. glabrata called G898D Upc2A. Chromatin immunoprecipitation coupled with Next Generation Sequencing (ChIP-seq) demonstrated that wild-type Upc2A binding to target genes was strongly induced by fluconazole while G898D Upc2A bound similarly, irrespective of drug treatment. We also carried out RNA-seq analysis to determine the genes that were direct or indirect targets of Upc2A transcriptional control. In addition to the well-described ERG genes as Upc2A transcriptional targets, we found a large group of genes encoding components of the translational apparatus along with membrane proteins. These Upc2A-regulated membrane protein-encoding genes are often targets of the Pdr1 transcription factor, demonstrating the high degree of overlap between these two regulatory networks. Finally, we provide evidence that Upc2A impacts the Pdr1-Cdr1 system during the anaerobic response and also modulates resistance to caspofungin. These studies provide a new perspective of Upc2A as a master regulator of lipid and membrane protein biosynthesis.

Comprehensive transcriptomic and proteomic analyses identify intracellular targets for myriocin to induce Fusarium oxysporum f. sp. niveum cell death

Background Myriocin is a natural product with antifungal activity and is derived from Bacillus amyloliquefaciens LZN01. Our previous work demonstrated that myriocin can inhibit the growth of Fusarium oxysporum f. sp. niveum (Fon) by inducing membrane damage. In this study, the antifungal actions of myriocin against Fon were investigated with a focus on the effects of myriocin on intracellular molecules. Results Analysis of DNA binding and fluorescence spectra demonstrated that myriocin can interact with dsDNA from Fon cells. The intracellular-targeted mechanism of action was also supported by transcriptomic and proteomic analyses; a total of 2238 common differentially expressed genes (DEGs) were identified. The DEGs were further verified by RT-qPCR. Most of the DEGs were assigned metabolism and genetic information processing functions and were enriched in ribosome biogenesis in eukaryotes pathway. The expression of some genes and proteins in ribosome biogenesis in eukaryotes pathway was affected by myriocin, primarily the genes controlled by the C6 zinc cluster transcription factor family and the NFYA transcription factor. Myriocin influenced the posttranscriptional processing of gene products by triggering the main RI (retained intron) events of novel alternative splicing; myriocin targeted key genes (FOXG_09470) or proteins (RIOK2) in ribosome biogenesis in eukaryotes pathway, resulting in disordered translation. Conclusions In conclusion, myriocin was determined to exhibit activity against Fon by targeting intracellular molecules. The results of our study may help to elucidate the antifungal actions of myriocin against Fon.

Comprehensive Transcriptomic and Proteomic Analyses Identify Intracellular Targets for Myriocin to Induce Fusarium Oxysporum F. Sp. Niveum Cell Death

Background: Myriocin is a natural product with antifungal activity and is derived from Bacillus amyloliquefaciens LZN01. Our previous work demonstrated that myriocin can inhibit the growth of Fusarium oxysporum f. sp. niveum (Fon) by inducing membrane damage. In this study, the antifungal actions of myriocin against Fon were investigated with a focus on the effects of myriocin on intracellular molecules.Results: Analysis of DNA binding and fluorescence spectra demonstrated that myriocin can interact with dsDNA from Fon cells. The intracellular-targeted mechanism of action was also supported by transcriptomic and proteomic analyses; a total of 2238 common differentially expressed genes (DEGs) were identified. The DEGs were further verified by RT-qPCR. Most of the DEGs were assigned metabolism and genetic information processing functions and were enriched in ribosome biogenesis in eukaryotes pathway. The expression of some genes and proteins in ribosome biogenesis in eukaryotes pathway was affected by myriocin, primarily the genes controlled by the C6 zinc cluster transcription factor family and the NFYA transcription factor. Myriocin influenced the posttranscriptional processing of gene products by triggering the main RI (retained intron) events of novel alternative splicing; myriocin targeted key genes (FOXG_09470) or proteins (RIOK2) in ribosome biogenesis in eukaryotes pathway, resulting in disordered translation. Conclusions: In conclusion, myriocin was determined to exhibit activity against Fon by targeting intracellular molecules. The results of our study may help to elucidate the antifungal actions of myriocin against Fon.

Genome-wide characterization of the Zn(II)2Cys6 zinc cluster-encoding gene family in Pleurotus ostreatus and expression analyses of this family during developmental stages and under heat stress

Pleurotus ostreatus is one of the most widely cultivated mushrooms in China. The regulatory mechanisms of fruiting body formation and the response to heat stress in P. ostreatus are main research focuses. The Zn(II)2Cys6 family is one of the largest families of transcriptional factors and plays important roles in multiple biological processes in fungi. In this study, we identified 66 zinc cluster proteins in P. ostreatus (PoZCPs) through a genome-wide search. The PoZCPs were classified into 15 types according to their zinc cluster domain. Physical and chemical property analyses showed a huge diversity among the PoZCPs. Phylogenetic analysis of PoZCPs classified these proteins into six groups and conserved motif combinations and similar gene structures were observed in each group. The expression profiles of these PoZCP genes during different developmental stages and under heat stress were further investigated by RNA-sequencing (RNA-seq), revealing diverse expression patterns. A total of 13 PoZCPs that may participate in development or the heat stress response were selected for validation of their expression levels through real-time quantitative PCR (RT-qPCR) analysis, and some developmental stage-specific and heat stress-responsive candidates were identified. The findings contribute to our understanding of the roles and regulatory mechanisms of ZCPs in P. ostreatus.