Benomyl-induced development and cardiac toxicity in zebrafish embryos.

Benomyl is a highly effective broad-spectrum fungicide widely used worldwide to control vegetable, fruit, and oil crop diseases. However, the mechanism of its toxicity to aquatic organisms and humans remains unknown. In this study, zebrafish were used to determine the toxicity of benomyl. It was found to be highly toxic, with a 72-h post-fertilization (hpf) lethal concentration 50 (LC50) of 1.454 mg/L. Benomyl induced severe developmental toxicity, including shorter body length, slower heart rate, and a reduced yolk absorption rate. Benomyl also increased oxidative stress in zebrafish, especially in the heart and head, as well as increasing malondialdehyde (MDA) content and decreasing catalase (CAT) and superoxide dismutase (SOD) activities. This indicates that benomyl induced reactive oxygen species (ROS) production and cell membrane peroxidation in vivo. Acridine orange (AO) staining and apoptosis factor detection further indicated that benomyl induced apoptosis in zebrafish. Overall, these findings demonstrate that benomyl disrupts cellular homeostasis by activating oxidative stress in zebrafish, resulting in an imbalance of cardiac development-related gene expression and apoptosis, which causes severe developmental toxicity and cardiac dysfunction. This study evaluated the in vivo toxicity of benomyl, which is a potential threat to aquatic organisms and humans. Possible toxicity mechanisms are explored, providing a valuable reference for the safe use of benomyl.

Suppression of arbuscular mycorrhizal fungi increased lead uptake in maize leaves and loss via surface runoff and interflow from polluted farmland.

Arbuscular mycorrhizal fungi (AMF) are ubiquitous in farmland. But the knowledge on AMF impact on lead (Pb) migration in farmland is limited. A field experiment was conducted in the rainy season (May-October) for two years in a Pb-polluted farmland. Benomyl was used to specifically suppress the native AMF growth in the farmland. The effect of benomyl-induced AMF suppression on the Pb uptake in maize, and Pb loss via surface runoff and interflows (20 cm and 40 cm depth) from the farmland was investigated. The benomyl significantly inhibited the AMF growth, resulting in decreases in the colonization rate, spore number, and contents of total and easily extractable glomalin-related soil protein (GRSP); and promoted the Pb migration into maize shoots and mainly enriched in leaves. The particulate Pb accounted for 83.2%-90.6% of Pb loss via surface runoff, while the proportion of particulate Pb loss via interflow was decreased and the proportion of dissolved Pb loss increased with the increase of soil depth. The AMF suppression led to a decrease in dissolved Pb concentration and loss, but an increase in particulate Pb concentration and loss, and enhanced the total Pb loss via surface runoff and interflows. Moreover, significant or very significant negative correlations were observed between the AMF colonization rate in roots with the Pb uptake in leaves, and the content of easily extractable GRSP with the particulate Pb loss. These results indicated the native AMF contributed to immobilizing Pb in soil and inhibited its migration to crops and the surrounding environment.

The sesquiterpenoid valerenic acid protects neuronal cells from the detrimental effects of the fungicide benomyl on apoptosis and DNA oxidation.

BACKGROUND: Valerenic acid (VA), a sesquiterpenoid of the plant Valeriana officinalis, has attracted attention of the research community due to its potential positive role against neurodegenerative diseases induced by chemicals. However, the relevant evidence in the literature is scarce. Therefore, this study aimed to examine the putative protective role of VA on the toxic effects of the fungicide benomyl on SH-SY5Y neural cells. METHODS: Cell viability was determined via the MTT and NRU assays, DNA damage was assessed via comet assay and apoptosis was evaluated through the expression of relevant genes. RESULTS: According to the results, exposure of the cells to benomyl enhanced viability inhibition and promoted DNA damage and apoptosis since the expression levels of the genes coding for MAPK8, NF-kB, Bax, Caspase-9 and Caspase-3 were increased. Treatment of the cells with VA ameliorated these effects in a concentration dependent manner. CONCLUSION: It is concluded that the molecular mechanism through which benomyl exerts its toxic action appears to depend on DNA oxidation and apoptosis induction. Furthermore, VA, a plant-derived compound is a protective antioxidant against pesticide-induced toxicity. Therefore, herbs, extracts and compounds of plant origin could be used as nutritional supplements that back up the beneficial role of medicine in neurodegenerative diseases.

Transcriptional Response of Candida auris to the Mrr1 Inducers Methylglyoxal and Benomyl.

Candida auris is an urgent threat to human health due to its rapid spread in health care settings and its repeated development of multidrug resistance. Diseases that increase risk for C. auris infection, such as diabetes, kidney failure, or immunocompromising conditions, are associated with elevated levels of methylglyoxal (MG), a reactive dicarbonyl compound derived from several metabolic processes. In other Candida species, expression of MG reductase enzymes that catabolize and detoxify MG are controlled by Mrr1, a multidrug resistance-associated transcription factor, and MG induces Mrr1 activity. Here, we used transcriptomics and genetic assays to determine that C. auris MRR1a contributes to MG resistance, and that the main Mrr1a targets are an MG reductase and MDR1, which encodes a drug efflux protein. The C. auris Mrr1a regulon is smaller than Mrr1 regulons described in other species. In addition to MG, benomyl (BEN), a known Mrr1 stimulus, induces C. auris Mrr1 activity, and characterization of the MRR1a-dependent and -independent transcriptional responses revealed substantial overlap in genes that were differentially expressed in response to each compound. Additionally, we found that an MRR1 allele specific to one C. auris phylogenetic clade, clade III, encodes a hyperactive Mrr1 variant, and this activity correlated with higher MG resistance. C. auris MRR1a alleles were functional in Candida lusitaniae and were inducible by BEN, but not by MG, suggesting that the two Mrr1 inducers act via different mechanisms. Together, the data presented in this work contribute to the understanding of Mrr1 activity and MG resistance in C. auris. IMPORTANCE Candida auris is a fungal pathogen that has spread since its identification in 2009 and is of concern due to its high incidence of resistance against multiple classes of antifungal drugs. In other Candida species, the transcription factor Mrr1 plays a major role in resistance against azole antifungals and other toxins. More recently, Mrr1 has been recognized to contribute to resistance to methylglyoxal (MG), a toxic metabolic product that is often elevated in different disease states. MG can activate Mrr1 and its induction of Mdr1 which can protect against diverse challenges. The significance of this work lies in showing that MG is also an inducer of Mrr1 in C. auris, and that one of the major pathogenic C. auris lineages has an activating Mrr1 mutation that confers protection against MG.

The cytoskeleton influences the formation and distribution of eisosomes in Neurospora crassa.

Eisosomes are stable protein complexes at the plasma membrane, with punctate distributional patterns. Their formation and how their locations are determined remain unclear. The current study discovered that the formation and distribution of eisosomes are influenced by the cytoskeleton. Disassembly of either the F-actin or the microtubules leads to eisosome localization at hyphal tips of germinated macroconidia in Neurospora crassa, and treatment with a high concentration of the microtubule-inhibitor benomyl results in the production of filamentous eisosome patterns. The defect in the cytoskeleton caused by the disassembly of microtubules or F-actin leads to an increased formation of eisosomes.

Endo-ß-1,3-glucanase (GH16 Family) from Trichoderma harzianum Participates in Cell Wall Biogenesis but Is Not Essential for Antagonism Against Plant Pathogens.

Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. ß-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have ß-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-ß-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.

Two herbicides, two fungicides and spore-associated bacteria affect Funneliformis mosseae extraradical mycelium structural traits and viability.

The extraradical mycelium (ERM) produced by arbuscular mycorrhizal fungi is fundamental for the maintenance of biological fertility in agricultural soils, representing an important inoculum source, together with spores and mycorrhizal root fragments. Its viability and structural traits, such as density, extent and interconnectedness, which are positively correlated with the growth and nutrition of host plants, may be affected by different agronomic practices, including the use of pesticides and by different mycorrhizospheric communities. This work, carried out using a whole-plant experimental model system, showed that structural traits of ERM, such as length and density, were strongly decreased by the herbicides dicamba and glufosinolate and the fungicides benomyl and fenhexamid, while anastomosis frequency and hyphal branching were differentially modulated by singly inoculated mycorrhizospheric bacteria, depending on their identity.

The genetic architecture in Saccharomyces cerevisiae that contributes to variation in drug response to the antifungals benomyl and ketoconazole.

The genetic basis of variation in drug response was investigated in individual Saccharomyces cerevisiae strains that exhibited different susceptibility to two antifungal agents: benomyl and ketoconazole. Following dose-response screening of 25 strains, 4 were selected on the basis of resistance or sensitivity relative to the standard laboratory strain BY. UWOPS87-2421 and L-1374 were respectively resistant and sensitive to benomyl; DBVPG6044 and Y12 were respectively resistant and sensitive to ketoconazole. We used advanced intercross lines and next generation sequencing-bulk segregant analysis to characterise the quantitative trait loci (QTL) underpinning drug responses after drug selection. Drug response was controlled by multiple QTL, ranging from a minimum of 5 to a maximum of 60 loci, almost all of which were not the primary drug target. For each drug, the resistant and the sensitive strain exhibited a number of shared loci, but also had strain-specific QTL. In our analysis, it was possible to estimate genetic effect of QTL, and a number of those shared between resistant and sensitive strains exhibited variable effect on the response phenotype. Thus, drug responses arise as a result of different genetic architectures, depending on the genetic background of the individual strain in question.

Npa3/ScGpn1 carboxy-terminal tail is dispensable for cell viability and RNA polymerase II nuclear targeting but critical for microtubule stability and function.

Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.

Benomyl-induced effects of ORMDL3 overexpression via oxidative stress in human bronchial epithelial cells.

The respiratory system is a major site of exposure route during pesticide use. Although pesticide exposure is associated with chronic respiratory diseases including asthma, the underlying pathophysiological mechanism remains to be elucidated. In this study, we investigated the in vitro effects of benomyl-induced ORMDL3 overexpression on the toxicological mechanism using the human bronchial epithelial cell line 16HBE14o-. Benomyl increased reactive oxygen species and Ca2+ levels, and asthma-related ADAM33 and ORMDL3 expression in 16HBE14o- cells. Considering the change in Ca2+ level and protein expression, we focused on ORMDL3 to elucidate the mechanism of benomyl-induced asthma. Antioxidant treatment showed that benomyl-induced ORMDL3 and endoplasmic reticulum stress could be triggered by oxidative stress. Furthermore, ORMDL3 knockdown alleviated the effects of benomyl on intracellular Ca2+, and the expression of metalloproteinases, and proinflammatory cytokines involved in the pathogenesis of asthma. In conclusion, our results suggest that benomyl-induced ORMDL3 overexpression via oxidative stress might be a mechanism involved in asthma. Moreover, antioxidants and alleviating mechanisms that reduce ORMDL3 levels could serve as promising therapeutic targets for pesticide-induced asthma.

An in vitro study of the antifungal activity of Trichoderma virens 7b and a profile of its non-polar antifungal components released against Ganoderma boninense.

Ganoderma boninense is the causal agent of a devastating disease affecting oil palm in Southeast Asian countries. Basal stem rot (BSR) disease slowly rots the base of palms, which radically reduces productive lifespan of this lucrative crop. Previous reports have indicated the successful use of Trichoderma as biological control agent (BCA) against G. boninense and isolate T. virens 7b was selected based on its initial screening. This study attempts to decipher the mechanisms responsible for the inhibition of G. boninense by identifying and characterizing the chemical compounds as well as the physical mechanisms by T. virens 7b. Hexane extract of the isolate gave 62.60% ± 6.41 inhibition against G. boninense and observation under scanning electron microscope (SEM) detected severe mycelial deformation of the pathogen at the region of inhibition. Similar mycelia deformation of G. boninense was observed with a fungicide treatment, Benlate® indicating comparable fungicidal effect by T. virens 7b. Fraction 4 and 5 of hexane active fractions through preparative thin layer chromatography (P-TLC) was identified giving the best inhibition of the pathogen. These fractions comprised of ketones, alcohols, aldehydes, lactones, sesquiterpenes, monoterpenes, sulphides, and free fatty acids profiled through gas chromatography mass spectrometry detector (GC/MSD). A novel antifungal compound discovery of phenylethyl alcohol (PEA) by T. virens 7b is reported through this study. T. virens 7b also proved to be an active siderophore producer through chrome azurol S (CAS) agar assay. The study demonstrated the possible mechanisms involved and responsible in the successful inhibition of G. boninense.

Histone H3K4 methylation regulates deactivation of the spindle assembly checkpoint through direct binding of Mad2.

Histone H3 methylation on Lys4 (H3K4me) is associated with active gene transcription in all eukaryotes. In Saccharomyces cerevisiae, Set1 is the sole lysine methyltransferase required for mono-, di-, and trimethylation of this site. Although H3K4me3 is linked to gene expression, whether H3K4 methylation regulates other cellular processes, such as mitosis, is less clear. Here we show that both Set1 and H3K4 mutants display a benomyl resistance phenotype that requires components of the spindle assembly checkpoint (SAC), including Bub3 and Mad2. These proteins inhibit Cdc20, an activator of the anaphase-promoting complex/cyclosome (APC/C). Mutations in Cdc20 that block Mad2 interactions suppress the benomyl resistance of both set1 and H3K4 mutant cells. Furthermore, the HORMA domain in Mad2 directly binds H3, identifying a new histone H3 "reader" motif. Mad2 undergoes a conformational change important for execution of the SAC. We found that the closed (active) conformation of both yeast and human Mad2 is capable of binding methylated H3K4, but, in contrast, the open (inactive) Mad2 conformation limits interaction with methylated H3. Collectively, our data indicate that interactions between Mad2 and H3K4 regulate resolution of the SAC by limiting closed Mad2 availability for Cdc20 inhibition.

Foliar Application of Extract from an Azalomycin-Producing Streptomyces malaysiensis Strain MJM1968 Suppresses Yam Anthracnose Caused by Colletotrichum gloeosporioides.

Yam anthracnose caused by Colletotrichum gloeosporioides (C.g) is the most devastating disease of yam (Dioscorea sp.). In the present study, we evaluated the culture filtrate extract (CFE) of azalomycin-producing Streptomyces malaysiensis strain MJM1968 for the control of yam anthracnose. MJM1968 showed strong antagonistic activity against C.g in vitro. Furthermore, the MJM1968 CFE was tested for inhibition of spore germination in C.g, where it completely inhibited spore germination at a concentration of 50 µg/ml. To assess the in planta efficacy of the CFE and spores of MJM1968 against C.g, a detached leaf bioassay was conducted, which showed both the treatments suppressed anthracnose development on detached yam leaves. Furthermore, a greenhouse study was conducted to evaluate the CFE from MJM1968 as a fungicide for the control of yam anthracnose. The CFE non-treated plants showed a disease severity of >92% after 90 days of artificial inoculation with C.g, whereas the disease severity of CFE-treated and benomyl-treated yam plants was reduced to 26% and 15%, respectively, after 90 days. Analysis of the yam tubers from the CFE-treated and non-treated groups showed that tubers from the CFE-treated plants were larger than that of non-treated plants, which produced abnormal smaller tubers typical of anthracnose. This study demonstrated the utility of the CFE from S. malaysiensis strain MJM1968 as a biofungicide for the control of yam anthracnose.

Mechanism of Action of the Benzimidazole Fungicide on Fusarium graminearum: Interfering with Polymerization of Monomeric Tubulin But Not Polymerized Microtubule.

Tubulins are the proposed target of clinically relevant anticancer drugs, anthelmintic, and fungicide. ß2-tubulin of the plant pathogen Fusarium graminearum was considered as the target of benzimidazole compounds by homology modeling in our previous work. In this study, α1-, α2-, and ß2-tubulin of F. graminearum were produced in Escherichia coli. Three benzimidazole compounds (carbendazim, benomyl, and thiabendazole) interacted with the recombinant ß2-tubulin and reduced the maximum fluorescence intensity of 2 µM ß2-tubulin 47, 50, and 25%, respectively, at saturation of compound-tubulin complexes. Furthermore, carbendazim significantly inhibited the polymerization of α1-/ß2-tubulins and α2-/ß2-tubulins 90.9 ± 0.4 and 93.5 ± 0.05%, respectively, in vitro. A similar result appeared with benomyl on the polymerization of α1-/ß2-tubulins and α2-/ß2-tubulins at 89.9 ± 0.1% and 92.6 ± 1.2% inhibition ratios, respectively. In addition, thiabendazole inhibited 81.6 ± 1% polymerization of α1-/ß2-tubulins, whereas it had less effect on α2-/ß2-tubulin polymerization, with 20.1 ± 1.9% inhibition ratio. However, the three compounds cannot destabilize the polymerized microtubule. To illuminate the issue, mapping the carbendazim binding sites and ß/α subunit interface on ß/α-tubulin complexes by homology modeling showed that the two domains were closed to each other. Understanding the nature of the interaction between benzimidazole compounds and F. graminearum tubulin is fundamental for the development of tubulin-specific anti-F. graminearum compounds.

Elucidation of novel budding yeast separase mutants.

The mitotic separase cleaves Scc1 in cohesin to allow sister chromatids to separate from each other upon anaphase onset. Separase is also required for DNA damage repair. Here, we isolated and characterized 10 temperature-sensitive (ts) mutants of separase ESP1 in the budding yeast Saccharomyces cerevisiae. All mutants were defective in sister chromatid separation at the restricted temperature. Some esp1-ts mutants were hypersensitive to the microtubule poison benomyl and/or the DNA-damaging agent bleomycin. Overexpression of securin alleviated the growth defect in some esp1-ts mutants, whereas it rather exacerbated it in others. The Drosophila Pumilio homolog MPT5 was isolated as a high-dosage suppressor of esp1-ts cells. We discuss various features of separase based on these findings.

Isolation of a spontaneous cerulenin-resistant sake yeast with both high ethyl caproate-producing ability and normal checkpoint integrity.

In the brewing of high-quality sake such as Daiginjo-shu, the cerulenin-resistant sake yeast strains with high producing ability to the flavor component ethyl caproate have been used widely. Genetic stability of sake yeast would be important for the maintenance of both fermentation properties of yeast and quality of sake. In eukaryotes, checkpoint mechanisms ensure genetic stability. However, the integrity of these mechanisms in sake yeast has not been examined yet. Here, we investigated the checkpoint integrity of sake yeasts, and the results suggested that a currently used cerulenin-resistant sake yeast had a defect in spindle assembly checkpoint (SAC). We also isolated a spontaneous cerulenin-resistant sake yeast FAS2-G1250S mutant, G9CR, which showed both high ethyl caproate-producing ability and integrity/intactness of the checkpoint mechanisms. Further, morphological phenotypic robustness analysis by use of CalMorph supported the genetic stability of G9CR. Finally, we confirmed the high quality of sake from G9CR in an industrial sake brewing setting.

Antiandrogenic mechanisms of pesticides in human LNCaP prostate and H295R adrenocortical carcinoma cells.

Several pesticides suspected or known to have endocrine disrupting effects were screened for pro- or antiandrogenic properties by determining their effects on proliferation, prostatic-specific antigen (PSA) secretion and androgen receptor (AR) expression, and AR phosphorylation in androgen-dependent LNCaP human prostate cancer cells, as well as on the expression and catalytic activity of the enzyme CYP17 in H295R human adrenocortical carcinoma cells, an in vitro model of steroidogenesis. Effects on SRD5A gene expression were determined in both cell lines. Benomyl, vinclozolin, and prochloraz, but not atrazine, concentration dependently (1-30 µM) decreased dihydrotestosterone (DHT)-stimulated proliferation of LNCaP cells. All pesticides except atrazine decreased DHT-stimulated PSA secretion, AR nuclear accumulation, and AR phosphorylation on serines 81 and 213 in LNCaP cells. Benomyl and prochloraz, but not vinclozolin or atrazine, decreased levels of CYP17 gene and protein expression, as well as catalytic activity in H295R cells. In the case of prochloraz, some of these effects corresponded with cytotoxicity. H295R cells expressed AR protein and SRD5A1, but not SRD5A2 transcripts. SRD5A1 gene expression in H295R cells was increased by 10 nM DHT, whereas in LNCaP cells significant induction was observed by 0.1 nM DHT. AR protein expression in H295R cells was not increased by DHT. Vinclozolin decreased DHT-induced SRD5A1 gene expression in LNCaP, but not H295R cells, indicating a functional difference of AR between the cell lines. In conclusion, pesticides may exert antiandrogenic effects through several mechanisms that are cell type-specific, including AR antagonism and down-regulation or catalytic inhibition of androgen biosynthetic enzymes, such as CYP17 and SRD5A1.

Confirmation of the cellular targets of benomyl and rapamycin using next-generation sequencing of resistant mutants in S. cerevisiae.

Investigating the mechanisms of action (MOAs) of bioactive compounds and the deconvolution of their cellular targets is an important and challenging undertaking. Drug resistance in model organisms such as S. cerevisiae has long been a means for discovering drug targets and MOAs. Strains are selected for resistance to a drug of interest, and the resistance mutations can often be mapped to the drug's molecular target using classical genetic techniques. Here we demonstrate the use of next generation sequencing (NGS) to identify mutations that confer resistance to two well-characterized drugs, benomyl and rapamycin. Applying NGS to pools of drug-resistant mutants, we develop a simple system for ranking single nucleotide polymorphisms (SNPs) based on their prevalence in the pool, and for ranking genes based on the number of SNPs that they contain. We clearly identified the known targets of benomyl (TUB2) and rapamycin (FPR1) as the highest-ranking genes under this system. The highest-ranking SNPs corresponded to specific amino acid changes that are known to confer resistance to these drugs. We also found that by screening in a pdr1Δ null background strain that lacks a transcription factor regulating the expression of drug efflux pumps, and by pre-screening mutants in a panel of unrelated anti-fungal agents, we were able to mitigate against the selection of multi-drug resistance (MDR) mutants. We call our approach "Mutagenesis to Uncover Targets by deep Sequencing", or "MUTseq", and show through this proof-of-concept study its potential utility in characterizing MOAs and targets of novel compounds.

Application of a new dual localization-affinity purification tag reveals novel aspects of protein kinase biology in Aspergillus nidulans.

Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients of SIN activity promote asymmetric septation.

Enhanced fungicide resistance in Isaria fumosorosea following ionizing radiation-induced mutagenesis.

The application of entomopathogenic fungi such as Isaria fumosorosea to combat insect pests on plants is complicated by their sensitivity to commonly used fungicides. In this study, I. fumosorosea mutants with enhanced resistance to the fungicide benomyl were induced by irradiation using either ion beams or gamma rays, or a combination of the two. When grown on agar containing benomyl, mycelial growth was observed for five of the six mutant isolates at benomyl concentrations that were more than 2000-fold those observed for the wild-type isolate (EC50 : > 5000 mg L(-1) c.f. EC50 : 2.5 mg L(-1) for the wild-type isolate). The mutant isolates evaluated also showed enhanced resistance to other fungicides at recommended field application rates. No differences were observed at the ß-tubulin locus between the wild-type and the mutant isolates, suggesting that the enhanced benomyl resistance was not attributable to mutations in that gene. Ion beams and gamma rays are thus potentially useful tools for inducing beneficial fungal mutations and thereby improving the potential for application of entomopathogenic fungi as microbial control agents.