Heterozygous Strains Research Articles

Rapid Colorimetric Detection of Genome Evolution in SCRaMbLEd Synthetic Saccharomyces cerevisiae Strains.

Genome-scale engineering and custom synthetic genomes are reshaping the next generation of industrial yeast strains. The Cre-recombinase-mediated chromosomal rearrangement mechanism of designer synthetic Saccharomyces cerevisiae chromosomes, known as SCRaMbLE, is a powerful tool which allows rapid genome evolution upon command. This system is able to generate millions of novel genomes with potential valuable phenotypes, but the excessive loss of essential genes often results in poor growth or even the death of cells with useful phenotypes. In this study we expanded the versatility of SCRaMbLE to industrial strains, and evaluated different control measures to optimize genomic rearrangement, whilst limiting cell death. To achieve this, we have developed RED (rapid evolution detection), a simple colorimetric plate-assay procedure to rapidly quantify the degree of genomic rearrangements within a post-SCRaMbLE yeast population. RED-enabled semi-synthetic strains were mated with the haploid progeny of industrial yeast strains to produce stress-tolerant heterozygous diploid strains. Analysis of these heterozygous strains with the RED-assay, genome sequencing and custom bioinformatics scripts demonstrated a correlation between RED-assay frequencies and physical genomic rearrangements. Here we show that RED is a fast and effective method to evaluate the optimal SCRaMbLE induction times of different Cre-recombinase expression systems for the development of industrial strains.

Systematic Target Screening Revealed That Tif302 Could Be an Off-Target of the Antifungal Terbinafine in Fission Yeast.

We used a heterozygous gene deletion library of fission yeasts comprising all essential and non-essential genes for a microarray screening of target genes of the antifungal terbinafine, which inhibits ergosterol synthesis via the Erg1 enzyme. We identified 14 heterozygous strains corresponding to 10 non-essential [7 ribosomal-protein (RP) coding genes, spt7, spt20, and elp2] and 4 essential genes (tif302, rpl2501, rpl31, and erg1). Expectedly, their erg1 mRNA and protein levels had decreased compared to the control strain SP286. When we studied the action mechanism of the non-essential target genes using cognate haploid deletion strains, knockout of SAGA-subunit genes caused a down-regulation in erg1 transcription compared to the control strain ED668. However, knockout of RP genes conferred no susceptibility to ergosterol-targeting antifungals. Surprisingly, the RP genes participated in the erg1 transcription as components of repressor complexes as observed in a comparison analysis of the experimental ratio of erg1 mRNA. To understand the action mechanism of the interaction between the drug and the novel essential target genes, we performed isobologram assays with terbinafine and econazole (or cycloheximide). Terbinafine susceptibility of the tif302 heterozygous strain was attributed to both decreased erg1 mRNA levels and inhibition of translation. Moreover, Tif302 was required for efficacy of both terbinafine and cycloheximide. Based on a molecular modeling analysis, terbinafine could directly bind to Tif302 in yeasts, suggesting Tif302 as a potential off-target of terbinafine. In conclusion, this genome-wide screening system can be harnessed for the identification and characterization of target genes under any condition of interest.

A heterozygous hypomorphic mutation of Fanca causes impaired follicle development and subfertility in female mice.

Reduced fertility is a common clinical feature of the individuals with Fanconi anemia (FA), a rare autosomal recessive disorder due to deficiency in FA pathway during DNA repair. Our previous study reported that the heterozygous pathogenic variants in FANCA (Fanconi anemia complementation group A) induced premature ovarian insufficiency (POI). However, the genotype-phenotype correlation in POI caused by FANCA variants remains considerably uncertain. Herein, a heterozygous non-frameshift Fanca-mutated mouse strain (Fanca+/hypo) carrying a 9-bp deletion (c.3581del9, p.QEA1194-1196del) was generated. The mutant mice exhibited slightly decreased Fanca protein level in ovaries, suggesting the non-frameshift deletion mutant is hypomorphic. Female fertility test showed decreased number of litters, litter sizes and prolonged litter interval time in the female Fanca+/hypo mice compared to wild-type mice. Follicle counting revealed a consistent decreasing pattern of follicle numbers in Fanca+/hypo females compared to that in wild-type mice with aging. Furthermore, embryonic fibroblasts of Fanca+/hypo mice were hyper-responsive to Mitomycin C in vitro, demonstrating a partial loss of function of this hypomorphic Fanca mutant in DNA repair. Collectively, our experimental observations suggest that the hypomorphic Fanca allele is sufficient to reduce female fertility in mice, providing new insights into the genetic counseling of FANCA variants in subfertile women.

Effect of the Ala234Asp replacement in mitochondrial branched-chain amino acid aminotransferase on the production of BCAAs and fusel alcohols in yeast.

In the yeast Saccharomyces cerevisiae, the mitochondrial branched-chain amino acid (BCAA) aminotransferase Bat1 plays an important role in the synthesis of BCAAs (valine, leucine, and isoleucine). Our upcoming study (Large et al. bioRχiv. 10.1101/2020.06.26.166157, Large et al. 2020) will show that the heterozygous tetraploid beer yeast strain, Wyeast 1056, which natively has a variant causing one amino acid substitution of Ala234Asp in Bat1 on one of the four chromosomes, produced higher levels of BCAA-derived fusel alcohols in the brewer's wort medium than a derived strain lacking this mutation. Here, we investigated the physiological role of the A234D variant Bat1 in S. cerevisiae. Both bat1∆ and bat1A234D cells exhibited the same phenotypes relative to the wild-type Bat1 strain-namely, a repressive growth rate in the logarithmic phase; decreases in intracellular valine and leucine content in the logarithmic and stationary growth phases, respectively; an increase in fusel alcohol content in culture medium; and a decrease in the carbon dioxide productivity. These results indicate that amino acid change from Ala to Asp at position 234 led to a functional impairment of Bat1, although homology modeling suggests that Asp234 in the variant Bat1 did not inhibit enzymatic activity directly. KEY POINTS: • Yeast cells expressing Bat1A234D exhibited a slower growth phenotype. • The Val and Leu levels were decreased in yeast cells expressing Bat1A234D. • The A234D substitution causes a loss-of-function in Bat1. • The A234D substitution in Bat1 increased fusel alcohol production in yeast cells.

The PHO pathway regulates white-opaque switching and sexual mating in the human fungal pathogen Candida albicans.

The opportunistic fungal pathogen Candida albicans is able to switch among several morphological phenotypes in response to environmental changes. White-opaque transition is a typical phenotypic switching system involved in the regulation of pathogenesis and sexual reproduction in C. albicans. Under regular laboratory culture conditions, to undergo white-to-opaque switching, cells must first undergo homozygosis at the mating-type locus (MTLa/a or α/α) since the a1/α2 heterodimer represses the expression of the Wor1 master regulator of switching in MTLa/α heterozygous strains. In this study, we report the roles of the PHO pathway of phosphate metabolism in the regulation of white-opaque switching and sexual mating in C. albicans. We find that deletion of the PHO pathway genes PHO81, PHO80, PHO2, and PHO4 induces the opaque phenotype in MTLa/α heterozygous cells. Low concentrations of external phosphate are conducive for the opaque phenotype in both MTL homozygous and heterozygous strains. Moreover, phosphate starvation can also increase the mating efficiency in C. albicans. Consistently, the pho80/pho80 mutant mimics an artificial phosphate starvation state and mates efficiently at both lower and higher phosphate concentrations. Our study establishes a link between the PHO pathway and white-opaque epigenetic switching in C. albicans.

Comparative Analysis of Virulence Factors of Homozygous and Heterozygous Strains of Candida albicans Vaginal Isolates.

Although the epidemiology of pathogenic Candida species is changing due to invasive diseases, Candida albicans has become the common cause of human infections worldwide. Candida albicans is a diploid yeast with a mostly clonal mode of reproduction and without known complete sexual cycle. This species has two heterozygous and homozygous strains at hyphal wall protein 1 gene locus (hwp1). Little is known about virulence factors of these strains. The aim of this study was to evaluate the exoenzyme activity of heterozygous and homozygous C. albicans strains. A total of 60 stock Candida albicans species isolates, which consisted of 30 homozygous and 30 heterozygous strains, were used for exoenzyme activities. We used egg yolk agar, Sabouraud blood agar, and bovine serum albumin agar for evaluation of phospholipase, hemolysin, and proteinase activity, respectively. Homozygous strains of Candida albicans had more phospholipase and proteinase activity than heterozygous strains. However, there were no significant statistical differences between the two strains in the severity of exoenzymes production. Beta hemolysin activity was seen in 100% and 96.7% of the homozygous and heterozygous strains, respectively. The results of this study indicated that both of the strains exhibited exoenzyme activities in different ranges. There were no significant statistical differences in virulence factors between the homozygous and heterozygous strains.

The Absence of PDR16 Gene Restricts the Overexpression of CaSNQ2 Gene in the Presence of Fluconazole in Candida albicans.

In yeast, the PDR16 gene encodes one of the PITP proteins involved in lipid metabolism and is regarded as afactor involved in clinical azole resistance of fungal pathogens. In this study, we prepared Candida albicans CaPDR16/pdr16Δ and Capdr16Δ/Δ heterozygous and homozygous mutant strains and assessed their responses to different stresses. The CaPDR16 deletion strains exhibited increased susceptibility to antifungal azoles and acetic acid. The addition of Tween80 restored the growth of Capdr16 mutants in the presence of azoles. However, the PDR16 gene deletion has not remarkable influence on sterol profile or membrane properties (membrane potential, anisotropy) of Capdr16Δ and Capdr16Δ/Δ mutant cells. Changes in halotolerance of C. albicans pdr16 deletion mutants were not observed. Fluconazole treatment leads to increased expression of ERG genes both in the wild-type and Capdr16Δ and Capdr16Δ/Δ mutant cells, and the amount of ergosterol and its precursors remain comparable in all three strains tested. Fluconazole treatment induced the expression of ATP-binding cassette transporter gene CaSNQ2 and MFS transporter gene CaTPO3 in the wild-type strain but not in the Capdr16Δ and Capdr16Δ/Δ mutants. The expression of CaSNQ2 gene markedly increased also in cells treated with hydrogen peroxide irrespective of the presence of CaPdr16p. CaPDR16 gene thus belongs to genes whose presence is required for full induction of CaSNQ2 and CaTPO3 genes in the presence of fluconazole in C. albicans.

A New Mouse Strain to Study Motoneurons in Pompe Disease

Pompe disease is a lysosomal storage disorder resulting from a deficiency in the lysosomal enzyme acid α‐glucosidase (GAA). The most severe form of the disease is associated with complete or near complete absence of functional GAA which results in cardiorespiratory failure and death within the first year of life. The only approved treatment for Pompe disease is human recombinant GAA (enzyme replacement therapy (ERT)) which improves overall survival rates but the recombinant protein does not reach the central nervous system. Respiratory impairment in Pompe disease has historically been attributed to muscle weakness but an accumulation of studies of Pompe patients and animal models indicate that motoneuron pathology is functionally relevant. In an effort to better understand the role of motoneurons in Pompe disease, we used breeding strategies to create a new mouse model with motoneuron histopathology features seen in Pompe disease while also allowing easy visualization of motoneurons via a fluorescent tag. Chat‐cre mice (Jackson) were crossed with Gaa−/− mice (Taconic). The resulting heterozygous strain were crossed with eYFP+/+ mice (courtesy Dr. Brian Harfe). Further breeding resulted in homozygous mice which were Chat‐cre+/+/Gaa−/−/eYFP+/+. These mice were then crossed to a Flox‐Gaa+/+ (Taconic) to create a heterozygous strain (Chat‐cre+/−/Gaa+/−/eYFP+/−/Flox‐Gaa+/−). Further inbreeding produced mice that were Chat‐cre+/+/Gaa+/−/eYFP+/+/Flox‐Gaa+/+. The intent of this final cross was to produce mice in which alpha motoneurons express eYFP but do not express GAA. Histological evaluation of the brainstem and spinal cord of these mice showed robust eYFP expression in the brainstem including the hypoglossal motor nucleus, nucleus ambiguous, and facial motor nucleus. In the cervical spinal cord, alpha motoneurons throughout the ventral horn also had robust fluorescence. This included putative phrenic motoneurons as determined based on location and size. The robust eYFP expression was restricted to motoneurons which indicates that motoneurons are Gaa−/− (based on the breeding scheme). Ongoing studies are evaluating GAA protein expression in peripheral tissues, glycogen accumulation in motoneurons, and also brainstem and spinal cord motoneuron loss over the lifespan. In addition, the eYFP signal in motoneurons will permit rigorous evaluation of motoneuron transduction efficacy in gene therapy experiments.Support or Funding Information2R01HD052682‐11A1 (DDF/BJB)

The caspase-like Gpi8 subunit of Candida albicans GPI transamidase is a metal-dependent endopeptidase

GPI anchored proteins (GPI-APs) act at the frontiers of cells, decoding environmental cues and determining host-pathogen interactions in several lower eukaryotes. They are also essential for viability in lower eukaryotes. The GPI biosynthetic pathway begins at the ER and follows a roughly linear pathway to generate the complete precursor (CP) glycolipid. The GPI transamidase (GPIT) transfers this glycolipid to the C-terminal end of newly translated proteins after removing their GPI attachment signal sequence (SS). The GPIT subunit that cleaves SS is Gpi8, a protein with a conserved Cys/His catalytic dyad typical of cysteine proteases. A CaGPI8 heterozygous mutant accumulates CPs and has reduced cell surface GPI-APs. Using a simple cell-free assay, we demonstrate that the heterozygous CaGPI8 strain has low endopeptidase activity as well. The revertant strain is restored in all these phenotypes. CaGpi8 is also shown to be a metalloenzyme, whose protease activity is sensitive to agents that modify Cys/His residues.

Inheritance of lambda-cyhalothrin resistance, fitness costs and cross-resistance to other pyrethroids in soybean looper, Chrysodeixis includens (Lepidoptera: Noctuidae)

Soybean looper (SBL), Chrysodeixis includens (Lepidoptera: Noctuidae), is one of the main lepidopteran pests of soybean and cotton crops. In this study, we selected a strain of SBL with resistance to lambda-cyhalothrin and evaluated the inheritance, fitness costs and cross-resistance to deltamethrin and cypermethrin. In inheritance pattern studies, SBL larvae were exposed to technical grade insecticides in topical bioassays. To investigate fitness costs, SBL strains were reared on artificial diet without insecticide and the survival, development and reproductive rates were evaluated. Estimated LD50 values of lambda-cyhalothrin for the susceptible and resistant strains were 0.0015 and 0.11 μg a.i./larva, respectively, indicating a resistance ratio (RR) of 73.3-fold. The LD50 values of heterozygous were 0.016 and 0.017 μg a.i./larva, suggesting an autosomal inheritance of resistance. On lambda-cyhalothrin sprayed-leaves, the resistant strain showed >85% survival. By contrast, heterozygous and susceptible strains had <64 and 8% survival, respectively, indicating an incompletely dominant resistance against the field dose of lambda-cyhalothrin. The resistant strain also showed cross-resistance to deltamethrin (RR = 6.2-fold) and cypermethrin (RR = 22.5-fold). Fitness costs studies showed that resistant insects had 71 and 48% lower survival and reproductive rates, respectively, than the susceptible strain (62.8% survival). The inheritance patterns of lambda-cyhalothrin resistance in SBL was autosomal, incompletely dominant and associated with fitness costs. SBL had low cross-resistance to deltamethrin and cypermethrin, indicating the potential to develop resistance to other pirethroids. Therefore, to preserve the useful life of chemical control against SBL are required the development of multi-tactic resistance management programs along with a reduction in insecticides applied.

Integrative description of bisexual Paramacrobiotus experimentalis sp. nov. (Macrobiotidae) from republic of Madagascar (Africa) with microbiome analysis

In a moss samples collected on Madagascar two populations of Paramacrobiotus experimentalis sp. nov. were found. Paramacrobiotus experimentalis sp. nov. with the presence of a microplacoid and areolatus type of eggs is similar to Pam. danielae, Pam. garynahi, Pam. hapukuensis, Pam. peteri, Pam. rioplatensis and Pam. savai, but it differs from them by some morphological and morphometric characters of the eggs. The p-distance between two COI haplotypes of Pam. experimentalis sp. nov. was 0.17%. In turn, the ranges of uncorrected genetic p-distances of all Paramacrobiotus species available in GenBank was from 18.27% (for Pam. lachowskae) to 25.26% (for Pam. arduus) with an average distance of 20.67%. We also found that Pam. experimentalis sp. nov. is bisexual. This observation was congruent on three levels: (i) morphological – specimen size dimorphism; (ii) structural (primary sexual characteristics) – females have an unpaired ovary while males have an unpaired testis and (iii) molecular – heterozygous and homozygous strains of the ITS-2 marker. Although symbiotic associations of hosts with bacteria (including endosymbiotic bacteria) are common in nature and these interactions exert various effects on the evolution, biology and reproductive ecology of hosts, there is still very little information on the bacterial community associated with tardigrades. To fill this gap and characterise the bacterial community of Pam. experimentalis sp. nov. populations and microbiome of its microhabitat, high throughput sequencing of the V3-V4 hypervariable regions in the bacterial 16S rRNA gene fragment was performed. The obtained 16S rRNA gene sequences ranged from 92,665 to 131,163. In total, 135 operational taxonomic units (OTUs) were identified across the rarefied dataset. Overall, both Pam. experimentalis sp. nov. populations were dominated by OTUs ascribed to the phylum Proteobacteria (89–92%) and Firmicutes (6–7%). In the case of samples from tardigrades’ laboratory habitat, the most abundant bacterial phylum was Proteobacteria (51–90%) and Bacteroides (9–48%). In all compared microbiome profiles, only 16 of 137 OTUs were shared. We found also significant differences in beta diversity between the partly species-specific microbiome of Pam. experimentalis sp. nov. and its culturing environment. Two OTUs belonging to a putative bacterial endosymbiont were identified – Rickettsiales and Polynucleobacter. We also demonstrated that each bacterial community was rich in genes involved in membrane transport, amino acid metabolism, and carbohydrate metabolism.

Evaluation of biofilm formation in the homozygous and heterozygous strains of vaginal Candida albicans isolates.

Background and Purpose: Candida albicans is one of the most opportunistic yeasts around the world. This species has two heterozygous and homozygous strains at hyphal wall protein 1 (hwp1) gene locus. A simple method for the discrimination of these two strains is the amplification of HWP1 gene. Regarding this, the aim of this study was to discriminate C. albicans heterozygous and homozygous strains via the amplification of hwp1 gene and evaluation of biofilm formation between the strains.Materials and Methods:A total of 60 homozygous (n=30) and heterozygous (n=30) strains were discriminated among 126 C. albicans vaginal isolates by the amplification of HWP1 gene, using specific primers. The evaluation of biofilm formation was accomplished using the visual method.Results:According to the results, the homozygous and heterozygous strains produced one and two DNA fragments, respectively. The frequency of homozygous strains among the C. albicans vaginal isolates was 76.2%. Biofilm formation activity in the heterozygous strains was more than that in the homozygous strains. However, statistical analysis showed no significant difference between the strains in terms of biofilm formation.Conclusion:As the findings indicated, the frequency of the heterozygous strains in C. albicans was lower than that of the homozygous strains. Both of the strains could form biofilm in the different ranges of severity. High activity of biofilm formation in heterozygous strains may set the ground for its pathogenicity.

No cross-resistance between ChinNPV and chemical insecticides in Chrysodeixis includens (Lepidoptera: Noctuidae).

Chrysodeixis includens nucleopolyhedrovirus (ChinNPV: Baculoviridae: Alphabaculovirus) is an active ingredient of a biological-based insecticide (Chrysogen®) recommended against soybean looper (SBL), Chrysodeixis includens (Walker, [1858]), in soybean in Brazil. We investigated if SBL strains resistant to chemical insecticides are cross-resistant to the baculovirus ChinNPV. In droplet feeding bioassays, SBL strains resistant to lambda-cyhalothrin and teflubenzuron showed equivalent susceptibility to ChinNPV as heterozygous and susceptible strains, indicating no cross-resistance between ChinNPV and chemical insecticides in SBL. Therefore, the ChinNPV is a valuable new "mode-of-action" tool for SBL resistance management in Brazil.

Random Chromosome Partitioning in the Polyploid Bacterium Thermus thermophilus HB27.

Little is known about chromosome segregation in polyploid prokaryotes. In this study, whether stringent or variable chromosome segregation occurs in polyploid thermophilic bacterium Thermus thermophilus was analyzed. A stable heterozygous strain (HL01) containing two antibiotic resistance markers at one gene locus was generated. The inheritance of the two alleles in the progeny of the heterozygous strain was then followed. During incubation without selection pressure, the fraction of heterozygous cells decreased and that of homozygous cells increased, while the relative abundance of each allele in the whole population remained constant, suggesting chromosome segregation had experienced random event. Consistently, in comparison with Bacillus subtilis in which the sister chromosomes were segregated equally, the ratios of DNA content in two daughter cells of T. thermophilus had a broader distribution and a larger standard deviation, indicating that the DNA content in the two daughter cells was not always identical. Further, the protein homologs (i.e., ParA and MreB) which have been suggested to be involved in bacterial chromosome partitioning did not actively participate in the chromosome segregation in T. thermophilus. Therefore, it seems that protein-based chromosome segregation machineries are less critical for the polyploid T. thermophilus, and chromosome segregation in this bacterium are not stringently controlled but tend to be variable, and random segregation can occur.

Laboratory and field survival of Spodoptera frugiperda (Lepidoptera: Noctuidae) on Bt and non-Bt maize and its susceptibility to insecticides.

Field-evolved resistance of fall armyworm (FAW), Spodoptera frugiperda (Smith), has been reported to Bt maize technologies in Brazil. The control failures of FAW by Bt maize increased the use of insecticides for their control. However, no information is available on the interaction between resistant FAW and their response to insecticides. Here, we evaluated the survival of FAW strains on Bt and non-Bt maize in laboratory and field conditions, and their susceptibility to insecticides. In the laboratory, resistant FAW larvae reared on Bt and non-Bt maize showed a similar susceptibility to spinetoram (LC50 = 0.16-0.18 µg a.i. cm-2 ) and chlorfenapyr (LC50 = 0.17-0.20 µg a.i. cm-2 ). However, their susceptibility was lower than that of the susceptible strain reared on non-Bt maize: LC50 = 0.05 (spinetoram) and 0.08 (chlorfenapyr) µg a.i. cm-2 . In contrast, heterozygous strains had similar susceptibility to the susceptible strain. In field trials, no differences in FAW survival were detected between strains when the commercial dose of two insecticides was applied in Bt and non-Bt maize. FAW strains surviving on Bt and non-Bt maize, at the same development stage, have similar susceptibility to insecticides. The integrated pest management practices and insect resistance management importance of these results are discussed. © 2019 Society of Chemical Industry.

Lack of relevant cross-resistance to Bt insecticide XenTari in strains of Spodoptera frugiperda (J. E. Smith) resistant to Bt maize.

The resistance of fall armyworm, Spodoptera frugiperda, has been characterized to Cry and Vip3A proteins of Bacillus thuringiensis (Bt) expressed in maize in Brazil. Here, we investigate the cross-resistance to Bt-insecticide XenTari in selected fall armyworm strains resistant to Bt maize varieties. The LC50 of XenTari in neonates of resistant strains ranged from 0.28 to 0.68 µg a.i./cm2, while for the susceptible reference strain (Sus), LC50 was 0.21 µg a.i./cm2. This indicated a resistance ratio lower than 3.2-fold. A similar variation in susceptibility was detected in EC50 values, which ranged from 0.04 to 0.13 µg a.i./cm2, demonstrating a maximum resistance ratio of 4.3-fold relative to the Sus strain (EC50 = 0.03 µg a.i./cm2). In the F1 progeny from reciprocal crosses, the LC50 ranged from 0.28 to 0.64 µg a.i./cm2 and EC50 from 0.03 to 0.18 µg a.i./cm2, similar to the values verified in parental resistant strains and representing a maximum resistance ratio of 3.0 and 6.0-fold, respectively. We also determined that susceptibility of third instar larvae to XenTari decreased when compared to neonates, however the variation remained similar. For third instar larvae from resistant strains, LC50 of XenTari ranged from 10.79 to 39.85 µg a.i./cm2, while for the Sus strain, LC50 was 9.25 µg a.i./cm2 (resistance ratio inferior to 4.3-fold). At the same stage, in heterozygous strains the LC50 ranged from 14.75 to 58.47 µg a.i./cm2 (resistance ratio inferior to 6.3-fold). Our data demonstrate a lack of significant cross-resistance to Bt-based insecticide XenTari in fall armyworm strains with resistance to Bt maize varieties.

Fitness costs of Vip3A resistance in Spodoptera frugiperda on different hosts.

Fall armyworm, Spodoptera frugiperda, is a major target pest of the Vip3A protein. The fitness of Bacillus thuringiensis (Bt)-resistant insects on refuges is an important factor in determining the speed of resistance development. Fitness costs associated with Bt resistance can vary among host plants. Here, we provide the first experimental evaluation of the interactions between non-Bt hosts and fitness costs of Vip3A resistance in S. frugiperda. Laboratory bioassays showed that survivorship, pupal weight, sex ratio, developmental time, fecundity, net reproductive rate, and intrinsic rate were not different among Vip3A-resistant (RR), -susceptible (SS) and -heterozygous (R♂ S♀ and R♀ S♂ ) strains of S. frugiperda on non-Bt corn, non-Bt cotton, and non-Bt diet. R♂ S♀ and R♀ S♂ also showed no differences on sorghum relative to SS. However, compared to SS, RR on sorghum showed significant reduction in pupal weight, and took longer time to develop to pupa and adult. These findings suggest that recessive fitness costs of reduced pupal weight and growth rate were detected at the individual level in the Vip3A resistant populations of S. frugiperda on sorghum. However, at the population level in terms of net reproductive rate and intrinsic rate, fitness costs of Vip3A resistance was not evident in S. frugiperda. © 2018 Society of Chemical Industry.

CORT0C04210 is required for Candida orthopsilosis adhesion to human buccal cells

Candida orthopsilosis is a human fungal pathogen belonging to the Candida parapsilosis sensu lato species complex. C. orthopsilosis annotated genome harbors 3 putative agglutinin-like sequence (ALS) genes named CORT0B00800, CORT0C04210 and CORT0C04220. The aim of this study was to investigate the role played by CORT0C04210 (CoALS4210) in the virulence and pathogenicity of this opportunistic yeast. Heterozygous and null mutant strains lacking one or both copies of CoALS4210 were obtained using the SAT1-flipper cassette strategy and were characterized in in vitro, ex vivo and in vivo models. While no differences between the mutant and the wild-type strains were observed in in vitro growth or in the ability to undergo morphogenesis, the CoALS4210 null mutant showed an impaired adhesion to human buccal epithelial cells compared to heterozygous and wild type strains. When the pathogenicity of CoALS4210 mutant and wild type strains was evaluated in a murine model of systemic candidiasis, no statistically significant differences were observed in fungal burden of target organs. Since gene disruption could alter chromatin structure and influence transcriptional regulation of other genes, two independent CRISPR/Cas9 edited mutant strains were generated in the same genetic background used to create the deleted strains. CoALS4210-edited strains were tested for their in vitro growing ability, and compared with the deleted strain for adhesion ability to human buccal epithelial cells. The results obtained confirmed a reduction in the adhesion ability of C. orthopsilosis edited strains to buccal cells. These findings provide the first evidence that CRISPR/Cas9 can be successfully used in C. orthopsilosis and demonstrate that CoALS4210 plays a direct role in the adhesion of C. orthopsilosis to human buccal cells but is not primarily involved in the onset of disseminated candidiasis.

Draft Genome Sequence of a Highly Heterozygous Yeast Strain from the Metschnikowia pulcherrima Subclade, UCD127.

ABSTRACTMetschnikowia strain UCD127 was isolated from soil in Ireland and sequenced. It is a highly heterozygous diploid strain with 385,000 single nucleotide polymorphisms (SNPs). Its ribosomal DNA has the highest similarity to that of M. chrysoperlae, but its ACT1 and TEF1 loci and mitochondrial genome show affinity to those of M. fructicola, whose genome is significantly larger.

Heterozygous diploid and interspecies SCRaMbLEing

SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution) is a genome restructuring technique that can be used in synthetic genomes such as that of Sc2.0, the synthetic yeast genome, which contains hundreds to thousands of strategically positioned loxPsym sites. SCRaMbLE has been used to induce rearrangements in yeast strains harboring one or more synthetic chromosomes, as well as plasmid DNA in vitro and in vivo. Here we describe a collection of heterozygous diploid strains produced by mating haploid semisynthetic Sc2.0 strains to haploid native parental strains. We subsequently demonstrate that such heterozygous diploid strains are more robust to the effects of SCRaMbLE than haploid semisynthetic strains, rapidly improve rationally selected phenotypes in SCRaMbLEd heterozygous diploids, and establish that multiple sets of independent genomic rearrangements are able to lead to similar phenotype enhancements. Finally, we show that heterozygous diploid SCRaMbLE can also be carried out in interspecies hybrid strains.