Phase-separation antagonists potently inhibit transcription and broadly increase nucleosome density.

Biomolecular condensates are self-organized membraneless bodies involved in many critical cellular activities, including ribosome biogenesis, protein synthesis, and gene transcription. Aliphatic alcohols are commonly used to study biomolecular condensates, but their effects on transcription are unclear. Here, we explore the impact of the aliphatic dialcohol, 1,6-hexanediol (1,6-HD), on Pol II transcription and nucleosome occupancy in budding yeast. As expected, 1,6-HD, a reagent effective in disrupting biomolecular condensates, strongly suppressed the thermal stress-induced transcription of Heat Shock Factor 1-regulated genes that have previously been shown to physically interact and coalesce into intranuclear condensates. Surprisingly, the isomeric dialcohol, 2,5-HD, typically used as a negative control, abrogated Heat Shock Factor 1-target gene transcription under the same conditions. Each reagent also abolished the transcription of genes that do not detectably coalesce, including Msn2/Msn4-regulated heat-inducible genes and constitutively expressed housekeeping genes. Thus, at elevated temperature (39 °C), HDs potently inhibit the transcription of disparate genes and as demonstrated by chromatin immunoprecipitation do so by abolishing occupancy of RNA polymerase in chromatin. Concurrently, histone H3 density increased at least twofold within all gene coding and regulatory regions examined, including quiescent euchromatic loci, silent heterochromatic loci, and Pol III-transcribed loci. Our results offer a caveat for the use of HDs in studying the role of condensates in transcriptional control and provide evidence that exposure to these reagents elicits a widespread increase in nucleosome density and a concomitant loss of both Pol II and Pol III transcription.

Promoters adopt distinct dynamic manifestations depending on transcription factor context.

Cells respond to external signals and stresses by activating transcription factors (TF), which induce gene expression changes. Prior work suggests that signal-specific gene expression changes are partly achieved because different gene promoters exhibit distinct induction dynamics in response to the same TF input signal. Here, using high-throughput quantitative single-cell measurements and a novel statistical method, we systematically analyzed transcriptional responses to a large number of dynamic TF inputs. In particular, we quantified the scaling behavior among different transcriptional features extracted from the measured trajectories such as the gene activation delay or duration of promoter activity. Surprisingly, we found that even the same gene promoter can exhibit qualitatively distinct induction and scaling behaviors when exposed to different dynamic TF contexts. While it was previously known that promoters fall into distinct classes, here we show that the same promoter can switch between different classes depending on context. Thus, promoters can adopt context-dependent "manifestations". Our analysis suggests that the full complexity of signal processing by genetic circuits may be significantly underestimated when studied in only specific contexts.

The InsP7 phosphatase Siw14 regulates inositol pyrophosphate levels to control localization of the general stress response transcription factor Msn2.

The environmental stress response (ESR) is critical for cell survival. Yeast cells unable to synthesize inositol pyrophosphates (PP-InsPs) are unable to induce the ESR. We recently discovered a diphosphoinositol pentakisphosphate (PP-InsP5) phosphatase in Saccharomyces cerevisiae encoded by SIW14 Yeast strains deleted for SIW14 have increased levels of PP-InsPs. We hypothesized that strains with high inositol pyrophosphate levels will have an increased stress response. We examined the response of the siw14Δ mutant to heat shock, nutrient limitation, osmotic stress, and oxidative treatment using cell growth assays and found increased resistance to each. Transcriptional responses to oxidative and osmotic stresses were assessed using microarray and reverse transcriptase quantitative PCR. The ESR was partially induced in the siw14Δ mutant strain, consistent with the increased stress resistance, and the mutant strain further induced the ESR in response to oxidative and osmotic stresses. The levels of PP-InsPs increased in WT cells under oxidative stress but not under hyperosmotic stress, and they were high and unchanging in the mutant. Phosphatase activity of Siw14 was inhibited by oxidation that was reversible. To determine how altered PP-InsP levels affect the ESR, we performed epistasis experiments with mutations in rpd3 and msn2/4 combined with siw14Δ. We show that mutations in msn2Δ and msn4Δ, but not rpd3, are epistatic to siw14Δ by assessing growth under oxidative stress conditions and expression of CTT1 Msn2-GFP nuclear localization was increased in the siw14Δ. These data support a model in which the modulation of PP-InsPs influence the ESR through general stress response transcription factors Msn2/4.

Mip6 binds directly to the Mex67 UBA domain to maintain low levels of Msn2/4 stress-dependent mRNAs.

RNA-binding proteins (RBPs) participate in all steps of gene expression, underscoring their potential as regulators of RNA homeostasis. We structurally and functionally characterize Mip6, a four-RNA recognition motif (RRM)-containing RBP, as a functional and physical interactor of the export factor Mex67. Mip6-RRM4 directly interacts with the ubiquitin-associated (UBA) domain of Mex67 through a loop containing tryptophan 442. Mip6 shuttles between the nucleus and the cytoplasm in a Mex67-dependent manner and concentrates in cytoplasmic foci under stress. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation experiments show preferential binding of Mip6 to mRNAs regulated by the stress-response Msn2/4 transcription factors. Consistent with this binding, MIP6 deletion affects their export and expression levels. Additionally, Mip6 interacts physically and/or functionally with proteins with a role in mRNA metabolism and transcription such as Rrp6, Xrn1, Sgf73, and Rpb1. These results reveal a novel role for Mip6 in the homeostasis of Msn2/4-dependent transcripts through its direct interaction with the Mex67 UBA domain.

Genetic screen of the yeast environmental stress response dynamics uncovers distinct regulatory phases.

Cells respond to environmental fluctuations by regulating multiple transcriptional programs. This response can be studied by measuring the effect of environmental changes on the transcriptome or the proteome of the cell at the end of the response. However, the dynamics of the response reflect the working of the regulatory mechanisms in action. Here, we utilized a fluorescent stress reporter gene to track the dynamics of protein production in yeast responding to environmental stress. The response is modulated by changes in both the duration and rate of transcription. We probed the underlying molecular pathways controlling these two dimensions using a library of ~1,600 single- and double-mutant strains. Dissection of the effects of these mutants and the interactions between them identified distinct modulators of response duration and response rate. Using a combination of mRNA-seq and live-cell microscopy, we uncover mechanisms by which Msn2/4, Mck1, Msn5, and the cAMP/PKA pathway modulate the response of a large module of stress-induced genes in two discrete regulatory phases. Our results and analysis show that transcriptional stress response is regulated by multiple mechanisms that overlap in time and cellular location.

Involvement of the stress-responsive transcription factor gene MSN2 in the control of amino acid uptake in Saccharomyces cerevisiae.

The transcriptional factor Msn2 plays a pivotal role in response to environmental stresses by activating the transcription of stress-responsive genes in Saccharomyces cerevisiae. Our previous studies demonstrate that intracellular proline acts as a key protectant against various stresses. It is unknown, however, whether Msn2 is involved in proline homeostasis in S. cerevisiae cells. We here found that MSN2-overexpressing (MSN2-OE) cells showed higher sensitivity to a toxic analogue of proline, l-azetidine-2-carboxylic acid (AZC), as well as to the other amino acid toxic analogues, than wild-type cells. Overexpression of MSN2 increased the intracellular content of AZC, suggesting that Msn2 positively regulates the uptake of proline. Among the known proline permease genes, GNP1 was shown to play a predominant role in the AZC toxicity. Based on quantitative real-time PCR and western blot analyses, the overexpression of MSN2 did not induce any increases in the transcript levels of GNP1 or the other proline permease genes, while the amount of the Gnp1 protein was markedly increased in MSN2-OE cells. Microscopic observation suggested that the endocytic degradation of Gnp1 was impaired in MSN2-OE cells. Thus, this study sheds light on a novel link between the Msn2-mediated global stress response and the amino acid homeostasis in S. cerevisiae.

The stress-regulatory transcription factors Msn2 and Msn4 regulate fatty acid oxidation in budding yeast.

The transcription factors Msn2 and Msn4 (multicopy suppressor of SNF1 mutation proteins 2 and 4) bind the stress-response element in gene promoters in the yeast Saccharomyces cerevisiae However, the roles of Msn2/4 in primary metabolic pathways such as fatty acid ß-oxidation are unclear. Here, in silico analysis revealed that the promoters of most genes involved in the biogenesis, function, and regulation of the peroxisome contain Msn2/4-binding sites. We also found that transcript levels of MSN2/MSN4 are increased in glucose-depletion conditions and that during growth in nonpreferred carbon sources, Msn2 is constantly localized to the nucleus in wild-type cells. Of note, the double mutant msn2Δmsn4Δ exhibited a severe growth defect when grown with oleic acid as the sole carbon source and had reduced transcript levels of major ß-oxidation genes. ChIP indicated that Msn2 has increased occupancy on the promoters of ß-oxidation genes in glucose-depleted conditions, and in vivo reporter gene analysis indicated reduced expression of these genes in msn2Δmsn4Δ cells. Moreover, mobility shift assays revealed that Msn4 binds ß-oxidation gene promoters. Immunofluorescence microscopy with anti-peroxisome membrane protein antibodies disclosed that the msn2Δmsn4Δ strain had fewer peroxisomes than the wild type, and lipid analysis indicated that the msn2Δmsn4Δ strain had increased triacylglycerol and steryl ester levels. Collectively, our data suggest that Msn2/Msn4 transcription factors activate expression of the genes involved in fatty acid oxidation. Because glucose sensing, signaling, and fatty acid ß-oxidation pathways are evolutionarily conserved throughout eukaryotes, the msn2Δmsn4Δ strain could therefore be a good model system for further study of these critical processes.

Stress response factors drive regrowth of quiescent cells.

Quiescent cells exploit an array of transcription factors to activate stress response machinery and maintain survival under nutrient-limited conditions. Our recent findings reveal that these transcription factors also play an important role in the exit of quiescence and regrowth. By studying Saccharomyces cerevisiae under a continuous, nutrient-limited condition, we found that Msn2 and Msn4 function as master regulators of glycolytic genes in the quiescent-like phase. They control the timing of transition from quiescence to growth by regulating the accumulation rate of acetyl-CoA, a key metabolite that is downstream of glycolysis and drives growth. These findings suggest a model that Msn2/4 not only protect the cells from starvation but also facilitate their regrowth from quiescence. Thus, understanding the functions of stress response transcription factors in metabolic regulation will provide deeper insight into how quiescent cells manage the capacity of regrowth.

Mechanisms for the epigenetic inheritance of stress response in single cells.

Cells have evolved to dynamically respond to different types of environmental and physiological stress conditions. The information about a previous stress stimulus experience by a mother cell can be passed to its descendants, allowing them to better adapt to and survive in new environments. In recent years, live-cell imaging combined with cell-lineage tracking approaches has elucidated many important principles that guide stress inheritance at the single-cell and population level. In this review, we summarize different strategies that cells can employ to pass the 'memory' of previous stress responses to their descendants. Among these strategies, we focus on a recent discovery of how specific features of Msn2 nucleo-cytoplasmic shuttling dynamics could be inherited across cell lineages. We also discuss how stress response can be transmitted to progenies through changes in chromatin and through partitioning of anti-stress factors and/or damaged macromolecules between mother and daughter cells during cell division. Finally, we highlight how emergent technologies will help address open questions in the field.

The role of yeast m6A methyltransferase in peroxisomal fatty acid oxidation.

The precise and controlled regulation of gene expression at transcriptional and post-transcriptional levels is crucial for the eukaryotic cell survival and functions. In eukaryotes, more than 100 types of post-transcriptional RNA modifications have been identified. The N6-methyladenosine (m6A) modification in mRNA is among the most common post-transcriptional RNA modifications known in eukaryotic organisms, and the m6A RNA modification can regulate gene expression. The role of yeast m6A methyltransferase (Ime4) in meiosis, sporulation, triacylglycerol metabolism, vacuolar morphology, and mitochondrial functions has been reported. Stress triggers triacylglycerol accumulation as lipid droplets. Lipid droplets are physically connected to the different organelles such as endoplasmic reticulum, mitochondria, and peroxisomes. However, the physiological relevance of these physical interactions remains poorly understood. In yeast, peroxisome is the sole site of fatty acid ß-oxidation. The metabolic status of the cell readily governs the number and physiological function of peroxisomes. Under low-glucose or stationary-phase conditions, peroxisome biogenesis and proliferation increase in the cells. Therefore, we hypothesized a possible role of Ime4 in the peroxisomal functions. There is no report on the role of Ime4 in peroxisomal biology. Here, we report that IME4 gene deletion causes peroxisomal dysfunction under stationary-phase conditions in Saccharomyces cerevisiae; besides, the ime4Δ cells showed a significant decrease in the expression of the key genes involved in peroxisomal ß-oxidation compared to the wild-type cells. Therefore, identification and determination of the target genes of Ime4 that are directly involved in the peroxisomal biogenesis, morphology, and functions will pave the way to better understand the role of m6A methylation in peroxisomal biology.

Condition-specific genetic interaction maps reveal crosstalk between the cAMP/PKA and the HOG MAPK pathways in the activation of the general stress response.

Cells must quickly respond and efficiently adapt to environmental changes. The yeast Saccharomyces cerevisiae has multiple pathways that respond to specific environmental insults, as well as a generic stress response program. The later is regulated by two transcription factors, Msn2 and Msn4, that integrate information from upstream pathways to produce fast, tunable, and robust response to different environmental changes. To understand this integration, we employed a systematic approach to genetically dissect the contribution of various cellular pathways to Msn2/4 regulation under a range of stress and growth conditions. We established a high-throughput liquid handling and automated flow cytometry system and measured GFP levels in 68 single-knockout and 1,566 double-knockout strains that carry an HSP12-GFP allele as a reporter for Msn2/4 activity. Based on the expression of this Msn2/4 reporter in five different conditions, we identified numerous genetic and epistatic interactions between different components in the network upstream to Msn2/4. Our analysis gains new insights into the functional specialization of the RAS paralogs in the repression of stress response and identifies a three-way crosstalk between the Mediator complex, the HOG MAPK pathway, and the cAMP/PKA pathway.

Identification of a transcription factor AoMsn2 of the Hog1 signaling pathway contributes to fungal growth, development and pathogenicity in Arthrobotrys oligospora.

INTRODUCTION: Arthrobotrys oligospora has been utilized as a model strain to study the interaction between fungi and nematodes owing to its ability to capture nematodes by developing specialized traps. A previous study showed that high-osmolarity glycerol (Hog1) signaling regulates the osmoregulation and nematocidal activity of A. oligospora. However, the function of downstream transcription factors of the Hog1 signaling in the nematode-trapping (NT) fungi remains unclear. OBJECTIVE: This study aimed to investigate the functions and potential regulatory network of AoMsn2, a downstream transcription factor of the Hog1 signaling pathway in A. oligospora. METHODS: The function of AoMsn2 was characterized using targeted gene deletion, phenotypic experiments, real-time quantitative PCR, RNA sequencing, untargeted metabolomics, and yeast two-hybrid analysis. RESULTS: Loss of Aomsn2 significantly enlarged and swollen the hyphae, with an increase in septa and a significant decrease in nuclei. In particular, spore yield, spore germination rate, traps, and nematode predation efficiency were remarkably decreased in the mutants. Phenotypic and transcriptomic analyses revealed that AoMsn2 is essential for fatty acid metabolism and autophagic pathways. Additionally, untargeted metabolomic analysis identified an important function of AoMsn2 in the modulation of secondary metabolites. Furtherly, we analyzed the protein interaction network of AoMsn2 based on the Kyoto Encyclopedia of Genes and Genomes pathway map and the online website STRING. Finally, Hog1 and six putative targeted proteins of AoMsn2 were identified by Y2H analysis. CONCLUSION: Our study reveals that AoMsn2 plays crucial roles in the growth, conidiation, trap development, fatty acid metabolism, and secondary metabolism, as well as establishes a broad basis for understanding the regulatory mechanisms of trap morphogenesis and environmental adaptation in NT fungi.

Deletion of the transcription factors Hsf1, Msn2 and Msn4 in yeast uncovers transcriptional reprogramming in response to proteotoxic stress.

The response to proteotoxic stresses such as heat shock allows organisms to maintain protein homeostasis under changing environmental conditions. We asked what happens if an organism can no longer react to cytosolic proteotoxic stress. To test this, we deleted or depleted, either individually or in combination, the stress-responsive transcription factors Msn2, Msn4, and Hsf1 in Saccharomyces cerevisiae. Our study reveals a combination of survival strategies, which together protect essential proteins. Msn2 and 4 broadly reprogram transcription, triggering the response to oxidative stress, as well as biosynthesis of the protective sugar trehalose and glycolytic enzymes, while Hsf1 mainly induces the synthesis of molecular chaperones and reverses the transcriptional response upon prolonged mild heat stress (adaptation).

PKA-Msn2/4-Shy1 cascade controls inhibition of proline utilization under wine fermentation models.

Proline, which is a predominant amino acid in grape musts, is involved in the taste and flavor of foods and beverages. The yeast Saccharomyces cerevisiae poorly utilizes proline in wine-making processes, leading to a nitrogen deficiency during fermentation and proline accumulation in wine. Previous studies have shown that the protein kinase A (PKA) pathway is involved in inhibitory mechanisms of proline utilization. In this study, we screened the PKA pathway-related genes that regulate proline utilization. Using a yeast culture collection of disrupted strains associated with the downstream of the PKA cascade, we revealed that the stress-responsive transcription factor genes MSN2/4 regulate proline utilization. Moreover, we found that Msn2/4 up-regulate the SHY1 gene during the cell growth of the wine fermentation model, which may cause the inhibition of proline utilization. The SHY1-deleted strain of the commercial wine yeast clearly showed proline consumption and average ethanol production under the wine fermentation model. The present data indicate that the PKA-Msn2/4-Shy1 cascade controls the inhibition of proline utilization under wine-making processes. Our study could hold promise for the development of wine yeast strains that can efficiently reduce proline during wine fermentation.

Involvement of the Sch9/Rim15/Msn2 signaling pathway in the anti-aging activity of dendrobine from Dendrobium nobile Lindl. via modification of oxidative stress and autophagy.

BACKGROUND: Aging is an important pathogenic factor of age-related diseases and has brought huge health threat and economic burden to the society. Dendrobium nobile Lindl., a valuable herb in China, promotes longevity according to the record of ancient Chinese materia medica. This study aimed to discover the material basis of D. nobile as an anti-aging herb and elucidate its action mechanism. METHODS: K6001 yeast replicative lifespan assay was used to guide the isolation of D. nobile. The chronological lifespan assay of YOM36 yeast was further conducted to confirm the anti-aging activity of dendrobine. The mechanism in which dendrobine exerts anti-aging effect was determined by conducting anti-oxidative stress assay, quantitative real-time PCR, Western blot, measurements of anti-oxidant enzymes activities, determination of nuclear translocation of Rim15 and Msn2, and replicative lifespan assays of Δsod1, Δsod2, Δcat, Δgpx, Δatg2, Δatg32, and Δrim15 yeasts. RESULTS: Under the guidance of K6001 yeast replicative lifespan system, dendrobine with anti-aging effect was isolated from D. nobile. The replicative and chronological lifespans of yeast were extended upon dendrobine treatment. In the study of action mechanism, dendrobine improved the survival rate of yeast under oxidative stress, decreased the levels of reactive oxygen species and malondialdehyde, and enhanced the enzyme activities and gene expression of superoxide dismutase and catalase, but it failed to elongate the replicative lifespans of Δsod1, Δsod2, Δcat, and Δgpx yeast mutants. Meanwhile, dendrobine enhanced autophagy occurrence in yeast but had no effect on the replicative lifespans of Δatg2 and Δatg32 yeast mutants. Moreover, the inhibition of Sch9 phosphorylation and the promotion of nuclear translocation of Rim15 and Msn2 were observed after treatment with denrobine. However, the effect of dendrobine disappeared from the Δrim15 yeast mutant after lifespan extension, oxidative stress reduction, and autophagy enhancement. CONCLUSIONS: Dendrobine exerts anti-aging activity in yeast via the modification of oxidative stress and autophagy through the Sch9/Rim15/Msn2 signaling pathway. Our work provides a scientific basis for the exploitation of D. nobile as an anti-aging herb.

Ddp1 Cooperates with Ppx1 to Counter a Stress Response Initiated by Nonvacuolar Polyphosphate.

In diverse cells from bacterial to mammalian species, inorganic phosphate is stored in long chains called polyphosphate (polyP). These nearly universal polymers, ranging from three to thousands of phosphate moieties in length, are associated with molecular functions, including energy homeostasis, protein folding, and cell signaling. In many cell types, polyphosphate is concentrated in subcellular compartments or organelles. In the budding yeast Saccharomyces cerevisiae, polyP synthesis by the membrane-bound vacuolar transporter chaperone (VTC) complex is coupled to its translocation into the lumen of the vacuole, a lysosome-like organelle, where it is stored at high concentrations. In contrast, the ectopic expression of the bacterial polyphosphate kinase (PPK) results in the toxic accumulation of polyP outside the vacuole. In this study, we used label-free mass spectrometry to investigate the mechanisms underlying this toxicity. We find that PPK expression results in the activation of a stress response mediated in part by the Hog1 and Yak1 kinases and the Msn2/Msn4 transcription factors as well as by changes in protein kinase A (PKA) activity. This response is countered by the combined action of the Ddp1 and Ppx1 polyphosphatases that function together to counter polyP accumulation and downstream toxicity. In contrast, the ectopic expression of previously proposed mammalian polyphosphatases did not impact PPK-mediated toxicity in this model, suggesting either that these enzymes do not function directly as polyphosphatases in vivo or that they require cofactors unique to higher eukaryotes. Our work provides insight into why polyP accumulation outside lysosome-like organelles is toxic. Furthermore, it serves as a resource for exploring how polyP may impact conserved biological processes at a molecular level. IMPORTANCE Cells from bacteria to humans have a molecule called polyphosphate (polyP) that functions in diverse processes. In many microbes, polyP is sequestered in granules or lysosome-related organelles such as vacuoles. In this study, we use an ectopic expression system to force budding yeast to accumulate polyP outside the vacuole. We use proteomics to demonstrate that this nonvacuolar polyP initiates a stress response mediated by a signaling cascade involving the Yak1 and Hog1 kinases and the Msn2 and Msn4 transcription factors. This response is countered by a pair of polyphosphatases with different enzymatic activities that function in concert to degrade polyP. Our results provide new insights into why polyP is confined to specific cell locations in many microbial cells.

cAMP-PKA and HOG1 signaling pathways regulate liamocin production by different ways via the transcriptional activator Msn2 in Aureobasidium melanogenum.

Liamocins, as the secondary metabolites synthesized and secreted by Aureobasidium spp., consist of a single mannitol or a single arabitol head group partially O-acylated with three 3,5-dihydroxydecanoic ester groups or directly esterified with three or four 3,5-dihydroxydecanoic ester tails. Very recently, the whole synthetic pathway of liamocins in A. melanogenum 6-1-2 has been elucidated. It was found that the promoter sequences of all the genes related to liamocin synthesis in A. melanogenum 6-1-2 had stress regulatory elements with core sequences of AGGGG or CCCCT. Therefore, expression of all the genes would be regulated by the Msn2. In this study, it was found that removal of the single one MSN2 gene in A. melanogenum 6-1-2 made the mutant decrease yield of extracellular liamocin by 92.28 %, while complementation of the MSN2 gene in the mutant rendered liamocin synthesis to be restored. When A. melanogenum 6-1-2 was cultured in the liamocin fermentation medium with high glucose and low nitrogen, the Msn2 was localized in the nucleus and positively regulated the expression of the genes related to liamocin biosynthesis. Furthermore, when the key BCY1 gene encoding regulatory subunit of the cAMP-PKA signaling pathway in A. melanogenum 6-1-2 was knocked out, the amount of extracellular liamocins synthesized by the mutant was decreased by 96.73 % and the Msn2 was localized in the cytoplasm. Similarly, when the key HOG1 gene in the HOG1 signaling pathway was deleted, liamocin biosynthesis in the knockout strain was decreased by 98.09 %. However, it was found that the Hog1 may be one part of the general transcription complex to regulate the transcription of the MSN2 gene, leading to the reduced Msn2 and liamocin synthesis in the mutant. In addition, the key TOR1 gene and SNF1 gene in the TOR1 signaling pathway and the SNF1 signaling pathway were not involved in the regulation of the Msn2 activity and liamocin synthesis. It was concluded that the transcriptional activator Msn2, the HOG1 signaling pathway and the cAMP-PKA signaling pathway were involved in the regulation of liamocin biosynthesis and production.

Transcription Factor MaMsn2 Regulates Conidiation Pattern Shift under the Control of MaH1 through Homeobox Domain in Metarhizium acridum.

The growth pattern of filamentous fungi can switch between hyphal radial polar growth and non-polar yeast-like cell growth depending on the environmental conditions. Asexual conidiation after radial polar growth is called normal conidiation (NC), while yeast-like cell growth is called microcycle conidiation (MC). Previous research found that the disruption of MaH1 in Metarhizium acridum led to a conidiation shift from NC to MC. However, the regulation mechanism is not clear. Here, we found MaMsn2, an Msn2 homologous gene in M. acridum, was greatly downregulated when MaH1 was disrupted (ΔMaH1). Loss of MaMsn2 also caused a conidiation shift from NC to MC on a nutrient-rich medium. Yeast one-hybrid (Y1H) and electrophoretic mobility shift assay (EMSA) showed that MaH1 could bind to the promoter region of the MaMsn2 gene. Disrupting the interaction between MaH1 and the promoter region of MaMsn2 significantly downregulated the transcription level of MaMsn2, and the overexpression of MaMsn2 in ΔMaH1 could restore NC from MC of ΔMaH1. Our findings demonstrated that MaMsn2 played a role in maintaining the NC pattern directly under the control of MaH1, which revealed the molecular mechanisms that regulated the conidiation pattern shift in filamentous fungi for the first time.

The Msn2 Transcription Factor Regulates Acaricidal Virulence in the Fungal Pathogen Beauveria bassiana.

Beauveria bassiana holds promise as a feasible biological control agent for tick control. The B. bassiana stress-response transcription factor Msn2 is known to contribute to fungal growth, conidiogenesis, stress-response and virulence towards insects; however, little is known concerning whether Msn2 is involved in infection across Arthropoda classes. We evaluated the effects of Msn2 on B. bassiana virulence against Rhipicephalus microplus (Acari, Ixodidae) using wild-type, targeted gene knockout (ΔBbmsn2) and complemented mutant (ΔBbmsn2/Bbmsn2) strains. Reproductive parameters of R. microplus engorged females treated topically or by an intra-hemocoel injection of conidial suspensions were assessed. Treated cuticles of engorged females were analyzed by microscopy, and proteolytic activity of B. bassiana on cuticles was assessed. Topically treated engorged females showed high mean larval hatching (>84%) in control and ΔBbmsn2 treatments, whereas treatment with the wild-type or ΔBbmsn2/Bbmsn2 strains resulted in significantly decreased (lowered egg viability) larval hatching. Percent control of R. microplus topically treated with ΔBbmsn2 was lower than in the groups treated with wild-type (56.1%) or ΔBbmsn2/Bbmsn2 strains. However, no differences on reproductive parameters were detected when R. microplus were treated by intra-hemocoel injection using low (800 conidia/tick) doses for all strains tested; R. microplus injected with high doses of wild-type or mutant strains (106 conidia/tick) died before laying eggs (~48 h after treatment). SEM analyses of B. bassiana infection showed similar conidial germination and formation of pseudo-appressoria on tick cuticle. Histological sections of ticks treated with the wild-type or ΔBbmsn2/Bbmsn2 strains showed fungal penetration through the cuticle, and into the tick interior. Hyphae of ΔBbmsn2, however, did not appear to penetrate or breach the tick exocuticle 120 h after treatment. Protease activity was lower on tick cuticles treated with ΔBbmsn2 than those treated with the wild-type or ΔBbmsn2/Bbmsn2 strains. These data show that loss of the Msn2 transcription factor reduced B. bassiana virulence against R. microplus, but did not interfere with conidial germination, appressoria formation or sporulation on tick cadavers, and plays only a minimal role once the cuticle is breached. Our results indicate that the BbMsn2 transcription factor acts mainly during the fungal penetration process and that decreased protease production may be one mechanism that contributes to the inability of the mutant strain to breach the tick cuticle.