Assessment of Fungal Lytic Enzymatic Extracts Produced Under Submerged Fermentation as Enhancers of Entomopathogens' Biological Activity.

The application of enzymes in agricultural fields has been little explored. One potential application of fungal lytic enzymes (chitinases, lipases, and proteases) is as an additive to current biopesticides to increase their efficacy and reduce the time of mortality. For this, a screening of lytic overproducer fungi under submerged fermentation with a chemical-defined medium was performed. Then, the enzymatic crude extract (ECE) was concentrated and partially characterized. This characterization consisted of measuring the enzymatic activity (lipase, protease and, chitinase) and determining the enzyme stability after storage at temperatures of - 80, - 20 and, 4 °C. And lastly, the application of these concentrated enzymatic crude extracts (C-ECE) as an enhancer of spores-based fungal biopesticide was proven. Beauveria were not as good producers of lytic enzymes as the strains from Trichoderma and Metarhizium. The isolate M. robertsii Mt015 was selected for the co-production of chitinases and proteases; and the isolate T. harzianum Th180 for co-production of chitinases, lipases, and proteases. The C-ECE of Mt015 had a protease activity of 18.6 ± 1.1 U ml-1, chitinase activity of 0.28 ± 0.01 U ml-1, and no lipase activity. Meanwhile, the C-ECE of Th180 reached a chitinase activity of 0.75 U ml-1, lipase activity of 0.32 U ml-1, and protease activity of 0.24 U ml-1. Finally, an enhancing effect of the enzymatic extracts of M. robertsii (66.7%) and T. harzianum (43.5%) on the efficacy of B. bassiana Bv064 against Diatraea saccharalis larvae was observed. This work demonstrates the non-species-specific enhancing effect of enzymatic extracts on the insecticidal activity of conidial-based biopesticides, which constitutes a contribution to the improvement of biological control agents' performance.

Plasma Promotes Fungal Cellulase Production by Regulating the Levels of Intracellular NO and Ca2.

For the industrial-scale production of useful enzymes by microorganisms, technological development is required for overcoming a technical bottleneck represented by poor efficiency in the induction of enzyme gene expression and secretion. In this study, we evaluated the potential of a non-thermal atmospheric pressure plasma jet to improve the production efficiency of cellulolytic enzymes in Neurospora crassa, a filamentous fungus. The total activity of cellulolytic enzymes and protein concentration were significantly increased (1.1~1.2 times) in media containing Avicel 24-72 h after 2 and 5 min of plasma treatment. The mRNA levels of four cellulolytic enzymes in fungal hyphae grown in media with Avicel were significantly increased (1.3~17 times) 2-4 h after a 5 min of plasma treatment. The levels of intracellular NO and Ca2+ were increased in plasma-treated fungal hyphae grown in Avicel media after 48 h, and the removal of intracellular NO decreased the activity of cellulolytic enzymes in media and the level of vesicles in fungal hyphae. Our data suggest that plasma treatment can promote the transcription and secretion of cellulolytic enzymes into the culture media in the presence of Avicel (induction condition) by enhancing the intracellular level of NO and Ca2+.

Aspergillosis in a colony of Humboldt penguins (Spheniscus humboldti) under managed care: a clinical and environmental investigation in a French zoological park.

Aspergillosis is pervasive in bird populations, especially those under human care. Its management can be critically impacted by exposure to high levels of conidia and by resistance to azole drugs. The fungal contamination in the environment of a Humboldt penguin (Spheniscus humboldti) group, housed in a French zoological park next to numerous large crop fields, was assessed through three serial sessions of surface sampling in nests, in 2018-20: all isolates were counted and characterized by sequencing. When identified as Aspergillus fumigatus, they were systematically screened for resistance mutations in the cyp51A gene and tested for minimal inhibitory concentrations (MICs) determination. At the same time, the clinical incidence of aspergillosis was evaluated in the penguin population by the means of systematic necropsy and mycological investigations. A microsatellite-based analysis tracked the circulation of A. fumigatus strains. Environmental investigations highlighted the substantial increase of the fungal load during the summer season (>12-fold vs. the other timepoints) and a large overrepresentation of species belonging to the Aspergillus section Fumigati, ranging from 22.7 to 94.6% relative prevalence. Only one cryptic species was detected (A. nishimurae), and one isolate exhibited G138S resistance mutation with elevated MICs. The overall incidence of aspergillosis was measured at ∼3.4% case-years, and mostly in juveniles. The analysis of microsatellite polymorphism revealed a high level of genetic diversity among A. fumigatus clinical isolates. In contrast, one environmental strain appeared largely overrepresented during the summer sampling session. In all, the rural location of the zoo did not influence the emergence of resistant strains.

A polyketide synthase from Verticillium dahliae modulates melanin biosynthesis and hyphal growth to promote virulence.

BACKGROUND: During the disease cycle, plant pathogenic fungi exhibit a morphological transition between hyphal growth (the phase of active infection) and the production of long-term survival structures that remain dormant during "overwintering." Verticillium dahliae is a major plant pathogen that produces heavily melanized microsclerotia (MS) that survive in the soil for 14 or more years. These MS are multicellular structures produced during the necrotrophic phase of the disease cycle. Polyketide synthases (PKSs) are responsible for catalyzing production of many secondary metabolites including melanin. While MS contribute to long-term survival, hyphal growth is key for infection and virulence, but the signaling mechanisms by which the pathogen maintains hyphal growth are unclear. RESULTS: We analyzed the VdPKSs that contain at least one conserved domain potentially involved in secondary metabolism (SM), and screened the effect of VdPKS deletions in the virulent strain AT13. Among the five VdPKSs whose deletion affected virulence on cotton, we found that VdPKS9 acted epistatically to the VdPKS1-associated melanin pathway to promote hyphal growth. The decreased hyphal growth in VdPKS9 mutants was accompanied by the up-regulation of melanin biosynthesis and MS formation. Overexpression of VdPKS9 transformed melanized hyphal-type (MH-type) into the albinistic hyaline hyphal-type (AH-type), and VdPKS9 was upregulated in the AH-type population, which also exhibited higher virulence than the MH-type. CONCLUSIONS: We show that VdPKS9 is a powerful negative regulator of both melanin biosynthesis and MS formation in V. dahliae. These findings provide insight into the mechanism of how plant pathogens promote their virulence by the maintenance of vegetative hyphal growth during infection and colonization of plant hosts, and may provide novel targets for the control of melanin-producing filamentous fungi.

Site-directed mutagenesis of the succinate dehydrogenase subunits B and D from Corynespora cassiicola reveals different fitness costs and sensitivities to succinate dehydrogenase inhibitors.

Carboxamide fungicides target succinate dehydrogenase (SDH). Recently published monitoring studies have shown that Corynespora cassiicola isolates are resistant to one or several SDH inhibitors (SDHIs) with amino acid substitutions in the SDH B and D subunits. We confirmed, by site-directed mutagenesis of the sdhB and sdhD genes, that each of the mutations identified in the field strains of C. cassiicola conferred resistance to boscalid and, in some cases, cross-resistance to other SDHIs (fluopyram, carboxin and penthiopyrad). Analyses of the enzyme activity and sdhB and sdhD gene expression show that modifications (SdhB_H278Y and SdhD_H105R) that result in a decline in SDH enzyme activity may be complemented by gene overexpression. The SdhB_H278Y, SdhB_I280V and SdhD_H105R mutants suffered large fitness penalties based on their biological properties, including conidia production and germination, mycelial growth, pathogenicity or survival abilities under environment stress. However, fitness cost was not found in the SdhB_H278R, SdhD_D95E and SdhD_G109V mutants. In the evaluation of resistance to boscalid in 2018 and 2019, the frequency of the SdhD_D95E and SdhD_G109V genotypes in the Liaoning and Shandong provinces changed dramatically compared with 2005-2017, from low resistance frequency (0.53% for D95E and 2.53% for G109V) to dominant resistance frequency (17.28% for D95E and 15.38% for G109V). Considering both the fitness and increased frequency of these genotypes, we may infer that the SdhD_D95E and SdhD_G109V mutants will be the dominant resistance mutants in field.

Assessment of the potential allergenicity and toxicity of Pichia proteins in a novel leghemoglobin preparation.

The production of soy leghemoglobin C2 (LegH) by Pichia pastoris (syn. K. phaffii) was developed by Impossible Foods to serve as a sustainable source of flavor and aroma in plant-based meats. The potential allergenicity and toxicity of a LegH from a new production process was analyzed using bioinformatics, proteomics and a pepsin digestion assay on leghemoglobin, and residual host proteins. LegH in the new preparation had the same proteoform as in the previous preparations as well as in soy root nodule extracts. Results of seven Pichia proteins, each representing ≥1% of the total protein content, showed no significant sequence matches to any known allergens with the exception of one, which matched the highly conserved wheat GAPDH, whose protein homolog is found in fungi and humans. Based on the data, it is unlikely that there is any risk of cross reactivity between LegH Prep and GAPDH. Pichia protein sequences showed very good alignment to homologous proteins from many common yeasts including Saccharomyces sp. In addition, LegH and Pichia proteins were all rapidly digested in a pepsin digest assay. In conclusion, LegH Prep from this P. pastoris production process is unlikely to pose a risk of food allergenicity.

Development of a loop-mediated isothermal amplification (LAMP) and a direct LAMP for the specific detection of Nosema ceranae, a parasite of honey bees.

Nosema ceranae is a ubiquitous microsporidian pathogen infecting the midgut of honey bees. The infection causes bee nosemosis, a disease associated with malnutrition, dysentery, and lethargic behavior, and results in considerable economic losses in apiculture. The use of a rapid, sensitive, and inexpensive DNA-based molecular detection method assists in the surveillance and eventual control of this pathogen. To this end, a loop-mediated isothermal amplification (LAMP) assay targeting the single-copy gene encoding the polar tube protein 3 (PTP3) has been developed. Genomic DNA of N. ceranae-infected forager bees sampled from distant geographic regions could be reliably amplified using the established LAMP assay. The N. ceranae-LAMP showed higher sensitivity than a classical reference PCR (98.6 vs 95.7%), when both approaches were applied to the detection of N. ceranae. LAMP detected a ten-fold lower infection rate than the reference PCR (1 pg vs 10 pg genomic DNA, respectively). In addition, we show highly specific and sensitive detection of N. ceranae from spore preparations in a direct LAMP format. No cross-reactions with genomic DNA and/or spores from N. apis, often co-infecting A. mellifera, or from N. bombi, infecting bumble bees, were observed. This low-cost and time-saving molecular detection method can be easily applied in simple laboratory settings, facilitating a rapid detection of N. ceranae in honey bees in epidemiological studies, surveillance and control, as well as evaluation of therapeutic measures against nosemosis.

Semi-pilot scale production of biodiesel from waste frying oil by genetically improved fungal lipases.

This paper addresses the issue of combining the usage of waste frying oil (WFO), as a feedstock, and a lipase produced in solid-state fermentation (SSF), as a biocatalyst, for semi-pilot scale production of biodiesel as fatty acid methyl esters (FAME). Two fungal mutants namely; Rhizopus stolonifer 1aNRC11 mutant F (1F) and Aspergillus tamarii NDA03a mutant G (3G) were used as a cocatalyst. The two mutants were cultivated separately by SSF in a tray bioreactor. The dried fermented solid of 1F and 3G mutants were used in a ratio of 3:1, respectively, for WFO transesterification. Optimization of several semi-pilot process stages including SSF and WFO transesterification reaction conditions resulted in 92.3% conversion of WFO to FAME. This FAME yield was obtained after 48 h using 10% cocatalyst (w/w of WFO), 10% water (w/w of WFO) and 3:1 methanol/ WFO molar ratio at 30 °C and 250 rpm. A preliminary economic evaluation of produced biodiesel price (190 $/Ton) is less than half the price of petroleum diesel in Egypt (401$/Ton) and is about 40.3% the price of biodiesel produced using a pure enzyme, which is a promising result. This strategy makes the biodiesel synthesis process greener, economical and sustainable.

Quantitative assessment of chaperone binding to amyloid aggregates identifies specificity of Hsp40 interaction with yeast prion fibrils.

Yeast self-perpetuating protein aggregates (yeast prions) provide a framework to investigate the interaction of misfolded proteins with the protein quality control machinery. The major component of this system that facilitates propagation of all known yeast amyloid prions is the Hsp104 chaperone that catalyzes fibril fragmentation. Overproduction of Hsp104 cures some yeast prions via a fragmentation-independent mechanism. Importantly, major cytosolic chaperones of the Hsp40 group, Sis1 and Ydj1, oppositely affect yeast prion propagation, and are capable of stimulating different activities of Hsp104. In this work, we developed a quantitative method to investigate the Hsp40 binding to amyloid aggregates. We demonstrate that Sis1 binds fibrils formed by the Sup35NM protein with higher affinity compared to Ydj1. Moreover, the interaction of Sis1 with the fibrils formed by the other yeast prion protein, Rnq1, is orders of magnitude weaker. We show that the deletion of the dimerization domain of Sis1 (crucial for the curing of [PSI+] by excess Hsp104) decreases its affinity to both Sup35NM and Rnq1 fibrils. Taken together, these results suggest that tight binding of Hsp40 to the amyloid fibrils is likely to enhance aggregate malpartition instead of fibril fragmentation.

Genomic analysis of a riboflavin-overproducing Ashbya gossypii mutant isolated by disparity mutagenesis.

BACKGROUND: Ashbya gossypii naturally overproduces riboflavin and has been utilized for industrial riboflavin production. To improve riboflavin production, various approaches have been developed. In this study, to investigate the change in metabolism of a riboflavin-overproducing mutant, namely, the W122032 strain (MT strain) that was isolated by disparity mutagenesis, genomic analysis was carried out. RESULTS: In the genomic analysis, 33 homozygous and 1377 heterozygous mutations in the coding sequences of the genome of MT strain were detected. Among these heterozygous mutations, the proportion of mutated reads in each gene was different, ranging from 21 to 75%. These results suggest that the MT strain may contain multiple nuclei containing different mutations. We tried to isolate haploid spores from the MT strain to prove its ploidy, but this strain did not sporulate under the conditions tested. Heterozygous mutations detected in genes which are important for sporulation likely contribute to the sporulation deficiency of the MT strain. Homozygous and heterozygous mutations were found in genes encoding enzymes involved in amino acid metabolism, the TCA cycle, purine and pyrimidine nucleotide metabolism and the DNA mismatch repair system. One homozygous mutation in AgILV2 gene encoding acetohydroxyacid synthase, which is also a flavoprotein in mitochondria, was found. Gene ontology (GO) enrichment analysis showed heterozygous mutations in all 22 DNA helicase genes and genes involved in oxidation-reduction process. CONCLUSION: This study suggests that oxidative stress and the aging of cells were involved in the riboflavin over-production in A. gossypii riboflavin over-producing mutant and provides new insights into riboflavin production in A. gossypii and the usefulness of disparity mutagenesis for the creation of new types of mutants for metabolic engineering.

Assessment of antifungal resistance and associated molecular mechanism in Candida albicans isolates from different cohorts of patients in North Indian state of Haryana.

The present study examines the trend in distribution of Candida species and their antifungal resistance patterns in hospitals across Haryana, a North Indian state with poorly addressed epidemiology of fungal infections. In our collection of 228 Candida isolates, Candida albicans dominated in both high vaginal swab (HVS) and urine samples while Candida glabrata and Candida tropicalis were the second-highest non-albicans Candida species (NAC), respectively. Of note, in blood samples, C. tropicalis and C. albicans were present in equal numbers. All 228 isolates were subjected to antifungal susceptibility tests, whereby 51% of C. albicans recovered from HVS samples displayed fluconazole resistance. To understand its mechanistic basis, expression profiling of efflux pump genes CDR1, CDR2, MDR1 and azole drug target, ERG11 was performed in 20 randomly selected resistant isolates, wherein many isolates elicited higher expression. Further, ERG11 gene sequencing suggested that most of the isolates harbored mutations, which are not reported with azole resistance. However, one isolate, RPCA9 (MIC 64 µg/mL) harbored triple mutation (Y132C, F145L, A114V), wherein Y132 and F145 sites were previously implicated in azole resistance. Interestingly, one isolate, (RPCA61) having MIC > 128 µg/mL harbored a novel mutation, G129R. Of note, HVS isolates RPCA 21, RPCA 22, and RPCA 44 (MICs 64 to > 128 µg/mL) did not show any change in alteration in ERG11 or overexpression of efflux pump genes. Together, this study presents a first report of Candida infections in selected hospitals of Haryana State.

Economical production of isomaltulose from agricultural residues in a system with sucrose isomerase displayed on Bacillus subtilis spores.

A safe, efficient, environmentally friendly process for producing isomaltulose is needed. Here, the biocatalyst, sucrose isomerase (SIase) from Erwinia rhapontici NX-5, displayed on the surface of Bacillus subtilis 168 spores (food-grade strain) was applied for isomaltulose production. The anchored SIase showed relatively high bioactivity, suggesting that the surface display system using CotX as the anchoring protein was successful. The stability of the anchored SIase was also significantly better. Thermal stability analysis showed that 80% of relative activity was retained after incubation at 40 °C and 45 °C for 60 min. To develop an economical industrial fermentation medium, untreated beet molasses (30 g/L) and cold-pressed soybean powder (50 g/L) were utilised as the main broth components for SIase pilot-scale production. Under the optimal conditions, the productive spores converted 92% of sucrose after 6 h and the conversion rate was 45% after six cycles. Isomaltulose production with this system using the agricultural residues, untreated beet molasses and soybean powder, as substrates is cost-effective and environmentally friendly and can help to overcome issues due to the genetic background.

Resistance to echinocandin antifungal agents in the United Kingdom in clinical isolates of Candida glabrata: Fifteen years of interpretation and assessment.

Candidemia is widely reported as the fourth most common form of bloodstream infection worldwide. Reports of breakthrough cases of candidemia are increasing, especially in the context of a move away from azole antifungals as prophylactic or first line treatment toward the use of echinocandin agents. The global evaluation of echinocandin antifungal susceptibility since 2003 has included switches in testing methodologies and the move to a sentinel echinocandin approach for classification reporting. This study compiles previously unpublished data from echinocandin susceptibility testing of UK clinical isolates of C. glabrata received at the Public Health England Mycology Reference Laboratory from 2003 to 2016 and reevaluates the prevalence of resistance in light of currently accepted testing protocols. From 2015 onward, FKS gene mutation detection using a novel Pyrosequencing® assay was assessed as a predictor of echinocandin resistance alongside conventional susceptibility testing. Overall, our data show that echinocandin resistance in UK isolates of C. glabrata is a rare phenomenon and prevalence has not appreciably increased in the last 14 years. The pyrosequencing assay was able to successfully detect hot spot mutations in FKS1 and FKS2, although not all isolates that exhibited phenotypic resistance demonstrated detectable hot spot mutations. We propose that a rapid genomic based detection method for FKS mutations, as part of a multifactorial approach to susceptibility testing, could help provide accurate and timely management decisions especially in regions where echinocandin resistance has been reported to be emerging in this important pathogen.

Advances in Rapid Molecular Blood Culture Diagnostics: Healthcare Impact, Laboratory Implications, and Multiplex Technologies.

BACKGROUND: For far too long, the diagnosis of bloodstream infections has relied on time-consuming blood cultures coupled with traditional organism identification and susceptibility testing. Technologies to define the culprit in bloodstream infections have gained sophistication in recent years, notably by application of molecular methods. CONTENT: In this review, we summarize the tests available to clinical laboratories for molecular rapid identification and resistance marker detection in blood culture bottles that have flagged positive. We explore the cost-benefit ratio of such assays, covering aspects that include performance characteristics, effect on patient care, and relevance to antibiotic stewardship initiatives. SUMMARY: Rapid blood culture diagnostics represent an advance in the care of patients with bloodstream infections, particularly those infected with resistant organisms. These diagnostics are relatively easy to implement and appear to have a positive cost-benefit balance, particularly when fully incorporated into a hospital's antimicrobial stewardship program.

Assessment of the in vitro and in vivo activity of atorvastatin against Candida albicans.

Aim. The aim of this work was to characterize the response of Candida albicans to atorvastatin, and to assess its in vivo antifungal capability.Methodology. The effect of atorvastatin on the growth and viability of C. albicans was assessed. The ability of the statin to alter cell permeability was quantified by measuring amino acid and protein leakage. The response of C. albicans to atorvastatin was assessed using label-free quantitative proteomics. The in vivo antifungal activity of atorvastatin was assessed using Galleria mellonella larvae infected with C. albicans.Results. Atorvastatin inhibited the growth of C. albicans. The atorvastatin-treated cells showed lower ergosterol levels than the controls, demonstrated increased calcofluor staining and released elevated quantities of amino acids and protein. Larvae infected with C. albicans showed a survival rate of 18.1±4.2 % at 144 h. In contrast, larvae administered atorvastatin (9.09 mg kg-1) displayed a survival rate of 60.2±6.4 % (P<0.05). Label-free quantitative proteomics identified 1575 proteins with 2 or more peptides and 465 proteins were differentially abundant (P<0.05). There was an increase in the abundance of enzymes with oxidoreductase and hydrolase activity in atorvastatin-treated cells, and squalene monooxygenase (4.52-fold increase) and lanosterol synthase (2.84-fold increase) were increased in abundance. Proteins such as small heat shock protein 21 (-6.33-fold) and glutathione peroxidase (-2.05-fold) were reduced in abundance.Conclusion. The results presented here indicate that atorvastatin inhibits the growth of C. albicans and is capable of increasing the survival of G. mellonella larvae infected with C. albicans.

Multifunctionalizing the marine diatom Phaeodactylum tricornutum for sustainable co-production of omega-3 long chain polyunsaturated fatty acids and recombinant phytase.

There is an urgent requirement for sustainable sources of food and feed due to world population growth. Aquaculture relies heavily on the fish meal and fish oils derived from capture fisheries, challenging sustainability of the production system. Furthermore, substitution of fish oil with vegetable oil and fish meal with plant seed meals in aquaculture feeds reduces the levels of valuable omega-3 long chain polyunsaturated fatty acids such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, and lowers the nutritional value due to the presence of phytate. Addition of exogenous phytase to fish feed is beneficial for enhancing animal health and reducing phosphorus pollution. We have engineered the marine diatom Phaeodactylum tricornutum, accumulating high levels of EPA and DHA together with recombinant proteins: the fungal Aspergillus niger PhyA or the bacterial Escherichia coli AppA phytases. The removal of the N-terminal signal peptide further increased phytase activity. Strains engineered with fcpA and CIP1 promoters showed the highest level of phytase activity. The best engineered strain achieved up to 40,000 phytase activity units (FTU) per gram of soluble protein, thus demonstrating the feasibility of development of multifunctionalized microalgae to simultaneously produce industrially useful proteins and fatty acids to meet the demand of intensive fish farming activity.

A simple and low cost tetra-primer ARMS-PCR method for detection triazole-resistant Aspergillus fumigatus.

The mutation at codon L98 accompanied by a tandem repeat of 34 base pairs (TR34/L98H) in the 5´upstream region of cyp51A is the principal mechanism of triazole resistance of Aspergillus fumigatus. We aimed to evaluate a simple and low-cost tetra-primer amplification refractory mutation system (ARMS)-PCR technique for detection of TR34/L98H mutations in the cyp51A gene of azole-resistant A. fumigatus. The tetra-primer ARMS-PCR assay optimized by four primers in one reaction consists of external primers for detection of tandem repeats in the promoter region and internal primers for detection of a point mutation in codon 98 (L98H) in the cyp51A gene of azole-resistant A. fumigatus. The specificity of TR34/L98H mutation detection was assessed by testing 36 clinical and environmental A. fumigatus strains. The tetra-primer ARMS-PCR assay from A. fumigatus, containing wild-type sequence (T allele) and L98H mutation at cyp51A (A allele), yielded two DNA fragments of 908 bp and 740 bp and two of 942 bp and 212 bp, respectively. None of the A. fumigatus isolates without the TR34/L98H mutation yielded false-positive results. The ARMS-PCR assay was 100% concordant with DNA sequencing results. Prevalence and screening of the TR34/L98H mutation in the cyp51A gene in A. fumigatus isolates may now be determined by a fast, low-cost, and simple method in resource-poor settings.

The fitness cost and benefit of phase-separated protein deposits.

Phase separation of soluble proteins into insoluble deposits is associated with numerous diseases. However, protein deposits can also function as membrane-less compartments for many cellular processes. What are the fitness costs and benefits of forming such deposits in different conditions? Using a model protein that phase-separates into deposits, we distinguish and quantify the fitness contribution due to the loss or gain of protein function and deposit formation in yeast. The environmental condition and the cellular demand for the protein function emerge as key determinants of fitness. Protein deposit formation can influence cell-to-cell variation in free protein abundance between individuals of a cell population (i.e., gene expression noise). This results in variable manifestation of protein function and a continuous range of phenotypes in a cell population, favoring survival of some individuals in certain environments. Thus, protein deposit formation by phase separation might be a mechanism to sense protein concentration in cells and to generate phenotypic variability. The selectable phenotypic variability, previously described for prions, could be a general property of proteins that can form phase-separated assemblies and may influence cell fitness.

Resistance risk assessment for fludioxonil in Sclerotinia homoeocarpa in China.

Sclerotinia homoeocarpa causes dollar spot disease on turfgrass and is a serious problem on many species worldwide. Fludioxonil, a phenylpyrrole fungicide, is not currently registered for dollar spot control in China. In this study, the baseline sensitivity to fludioxonil was established using an in vitro assay for 105 isolates of S. homoeocarpa collected from 10 locations in different regions of China. Results indicate that the frequency distribution of effective concentration for 50% inhibition of mycelial growth (EC50) values of the S. homoeocarpa isolates was unimodal (W = 0.9847, P = .2730). The mean EC50 value was 0.0020 ±â€¯0.0006 µg/ml with a range from 0.0003 to 0.0035 µg/ml. A total of 7 fludioxonil-resistant mutants were obtained in laboratory, the mutants were stable in fludioxonil sensitivity after the 10th transfer, with resistance factor (RF) ranging from 4.320 to >13,901.4. The mutants showed a positive cross-resistance between fludioxonil and the dicarboximide fungicide iprodione, but not propiconazole, fluazinam, and thiophanate-methyl. When mycelial growth rate, pathogenicity and osmotic sensitivity were assessed, the mutants decreased in the fitness compared with their parental isolates. Sequence alignment of the histidine kinase gene Shos1 revealed a 13-bp fragment deletion only in one mutant, no mutations were observed on Shos1 in the rest resistant mutants.

Allosteric mechanism of the circadian protein Vivid resolved through Markov state model and machine learning analysis.

The fungal circadian clock photoreceptor Vivid (VVD) contains a photosensitive allosteric light, oxygen, voltage (LOV) domain that undergoes a large N-terminal conformational change. The mechanism by which a blue-light driven covalent bond formation leads to a global conformational change remains unclear, which hinders the further development of VVD as an optogenetic tool. We answered this question through a novel computational platform integrating Markov state models, machine learning methods, and newly developed community analysis algorithms. Applying this new integrative approach, we provided a quantitative evaluation of the contribution from the covalent bond to the protein global conformational change, and proposed an atomistic allosteric mechanism leading to the discovery of the unexpected importance of A'α/Aß and previously overlooked Eα/Fα loops in the conformational change. This approach could be applicable to other allosteric proteins in general to provide interpretable atomistic representations of their otherwise elusive allosteric mechanisms.