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Abstract Inversions are balanced structural variants that often remain undetected in genetic diagnostics. We present a female proband with a de novo Chromosome 15 paracentric inversion, disrupting MEIS2 and NUSAP1. The inversion was detected by short-read genome sequencing and confirmed with adaptive long-read sequencing. The breakpoint junction analysis revealed a 96 bp (bp) deletion and an 18 bp insertion in the two junctions, suggesting that the rearrangement arose through a replicative error. Transcriptome sequencing of cultured fibroblasts revealed normal MEIS2 levels and 0.61-fold decreased expression of NUSAP1. Furthermore, three fusion transcripts were detected and confirmed by Sanger sequencing. Heterozygous loss of MEIS2 (MIM# 600987) is associated with a cleft palate, heart malformations, and intellectual impairment, which overlap with the clinical symptoms observed in the proband. The observed fusion transcripts are likely non-functional, and MEIS2 haploinsufficiency is the likely disease causative mechanism. Altogether, this study’s findings illustrate the importance of including inversions in rare disease diagnostic testing and highlight the value of long read sequencing for the validation and characterization of such variants.

Abstract CRISPR/Cas9 technology has been widely adopted for genome editing in a wide range of organisms, including many fungi. Pyricularia oryzae is a filamentous fungal pathogen that causes the devastating rice blast disease. However, an efficient and cost-effective CRISPR/Cas9 system for the rice blast fungus has yet to be established. Here, we report an 84-bp arginyl (Arg)-tRNA promoter-driven CRISPR/Cas9 system, which enables efficient and cost-effective gene editing in P. oryzae. Preliminary screening of three tRNAs from the 179 predicted tRNAs in P. oryzae showed that two Arg-tRNA CRISPR/Cas9 cassettes reproducibly generated MoB56 disruption efficiently. Further, five genes located on distinct chromosomes, including two previously uncharacterized genes, were randomly picked up to test the efficiency of the Mo_tRNAArg24-gRNA-Cas9 cassette. Ppg1 is a gene essential to the pathogenicity and important for mycelial growth and conidiation of P. oryzae, which is located at chromosome 2 and exhibited a relatively low gene replacement rate (

Inversions are balanced structural variants that often remain undetected in genetic diagnostics. We present a female proband with a de novo Chromosome 15 paracentric inversion, disrupting MEIS2 and NUSAP1. The inversion was detected by short-read genome sequencing and confirmed with adaptive long-read sequencing. The breakpoint junction analysis revealed a 96 bp (bp) deletion and an 18 bp insertion in the two junctions, suggesting that the rearrangement arose through a replicative error. Transcriptome sequencing of cultured fibroblasts revealed normal MEIS2 levels and 0.61-fold decreased expression of NUSAP1. Furthermore, three fusion transcripts were detected and confirmed by Sanger sequencing. Heterozygous loss of MEIS2 (MIM# 600987) is associated with a cleft palate, heart malformations, and intellectual impairment, which overlap with the clinical symptoms observed in the proband. The observed fusion transcripts are likely non-functional, and MEIS2 haploinsufficiency is the likely disease causative mechanism. Altogether, this study's findings illustrate the importance of including inversions in rare disease diagnostic testing and highlight the value of long read sequencing for the validation and characterization of such variants. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 technology has been widely adopted for genome editing in a wide range of organisms, including many fungi. Pyricularia oryzae is a filamentous fungal pathogen that causes the devastating rice blast disease. However, an efficient and cost-effective CRISPR/Cas9 system for the rice blast fungus has yet to be established. Here, we report an 84-bp arginyl (Arg)-tRNA promoter-driven CRISPR/Cas9 system, which enables efficient and cost-effective gene editing in P. oryzae. Preliminary screening of three tRNAs from the 179 predicted tRNAs in P. oryzae showed that two Arg-tRNA CRISPR/Cas9 cassettes reproducibly generated MoB56 disruption efficiently. Further, five genes located on distinct chromosomes, including two previously uncharacterized genes, were randomly picked up to test the efficiency of the Mo_tRNAArg24-gRNA-Cas9 cassette. Ppg1 is a gene essential to the pathogenicity and important for mycelial growth and conidiation of P. oryzae, which is located at chromosome 2 and exhibited a relatively low gene replacement rate (< 1/500) by the traditional gene replacement approach. By using the Mo_tRNAArg24-gRNA-Cas9 cassette, Ppg1 gene disruption rate was increased up to 75.9%. In addition, Bip2, an uncharacterized genes located close to the centromere of chromosome 4, was disrupted at 66.7%. For all the five tested genes, our Mo_tRNAArg24-gRNA-Cas9 cassette showed high gene disruption efficiency in P. oryzae, ranging from 66.7% to 100%. Importantly, it rarely induces Cas9 toxicity to P. oryzae. The Mo_tRNAArg24-gRNA-Cas9 cassette described in this study can be adopted as an alternative for functional genomics study in P. oryzae. [ABSTRACT FROM AUTHOR]

Fusarium verticillioides produces fumonisins, which are mycotoxins inhibiting sphingolipid biosynthesis in humans, animals, and other eukaryotes. Fumonisins are presumed virulence factors of plant pathogens, but may also play a role in interactions between competing fungi. We observed higher resistance to added fumonisin B1 (FB1) in fumonisin-producing Fusarium verticillioides than in nonproducing F. graminearum, and likewise between isolates of Aspergillus and Alternaria differing in production of sphinganine-analog toxins. It has been reported that in F. verticillioides, ceramide synthase encoded in the fumonisin biosynthetic gene cluster is responsible for self-resistance. We reinvestigated the role of FUM17 and FUM18 by generating a double mutant strain in a fum1 background. Nearly unchanged resistance to added FB1 was observed compared to the parental fum1 strain. A recently developed fumonisin-sensitive baker's yeast strain allowed for the testing of candidate ceramide synthases by heterologous expression. The overexpression of the yeast LAC1 gene, but not LAG1, increased fumonisin resistance. High-level resistance was conferred by FUM18, but not by FUM17. Likewise, strong resistance to FB1 was caused by overexpression of the presumed F. verticillioides "housekeeping" ceramide synthases CER1, CER2, and CER3, located outside the fumonisin cluster, indicating that F. verticillioides possesses a redundant set of insensitive targets as a self-resistance mechanism. [ABSTRACT FROM AUTHOR]

• Investigation of lncRNA FOXD2-AS1 in oral squamous cell carcinoma (OSCC):** our study explores the expression, prognostic significance, and functional roles of lncRNA FOXD2-AS1 in OSCC, providing novel insights into its involvement in cancer progression. • FOXD2-AS1 promotes OSCC Proliferation, Invasion, and Migration:** through comprehensive in vitro and in vivo analyses, we demonstrate that FOXD2-AS1 overexpression significantly enhances OSCC cell growth, invasion, and migration capabilities. • Regulatory mechanism via miR-185–5p/PLOD1/Akt/mTOR Pathway:** the study uncovers that FOXD2-AS1 modulates OSCC pathogenesis by targeting the miR-185–5p/PLOD1/Akt/mTOR signaling pathway, revealing a complex regulatory network influencing cancer cell behavior. • Prognostic potential of FOXD2-AS1 in OSCC:** bioinformatics and experimental data indicate that high FOXD2-AS1 expression correlates with poor prognosis in OSCC patients, suggesting its utility as a prognostic biomarker. • Therapeutic implications for OSCC Treatment:** identifying FOXD2-AS1 as a key regulator of OSCC progression opens new avenues for developing targeted therapies, potentially improving treatment outcomes for patients with this aggressive cancer type. • Nomogram model for survival Prediction:** a nomogram model incorporating FOXD2-AS1 expression was constructed to predict OSCC patient survival, offering a valuable tool for clinical prognosis assessment. Although lncRNAs are recognized to contribute to the development of oral squamous-cell carcinoma (OSCC), their exact function in invasion and cell migration is not clear. In this research, we explored the molecular and cellular mechanisms of FOXD2-AS1 in OSCC. Prognostic and bioinformatics analyses were used to test for the differential expression of FOXD2-AS1-PLOD1. Following FOXD2-AS1 suppression or overexpression, changes in cell viability were measured using the CCK-8 test; changes in cell migration and invasion abilities were measured using the migration and the Transwell assay. The expression of associated genes and proteins was found using Western blot and RT-qPCR. Analysis of luciferase reporter genes was done to look for regulatory connections between various molecules. The FOXD2-AS1-PLOD1 pair, which was highly expressed in OSCC, was analyzed and experimentally verified to be closely related to the prognosis of OSCC, and a nomogram model and correction curve were constructed. The inhibition of FOXD2-AS1 resulted in the reduction of cell activity, migration, invasion ability and changes in genes related to invasion and migration. In vivo validation showed that inhibition of FOXD2-AS1 expression slowed tumor growth, and related proteins changed accordingly. The experiments verified that FOXD2-AS1 negatively regulated miR-185–5 p and that miR-185–5 p negatively regulated PLOD1. In addition, it was found that the expression of PLOD1, p-Akt and p-mTOR proteins in OSCC cells was reduced by the inhibition of FOXD2-AS1, and FOXD2-AS1 and PLOD1 were closely related to the Akt/mTOR pathway. Increased expression of FOXD2-AS1 promotes OSCC growth, invasion and migration, which is important in part by targeting miR-185–5 p/PLOD1/Akt/mTOR pathway activity. [Display omitted] [ABSTRACT FROM AUTHOR]

Three new cyclic lipopeptides, olenamidonins A-C (1–3), in addition to two previously reported metabolites (4 and 5), were accumulated in the ΔdtxRso deletion mutant of deepsea-derived Streptomyces olivaceus SCSIO 1071. The structures of these cyclic lipopeptides were determined by a combination of spectroscopic methods and circular dichroism (CD) measurement. The antibacterial assay results showed that compounds 1–5 displayed different degrees of growth inhibition against multidrug-resistant (MDR) bacterial strains Enterococcus faecalis CCARM 5172 and Enterococcus faecium CCARM 5203 with minimum inhibitory concentrations (MICs) of 1.56−6.25 μg/mL. [ABSTRACT FROM AUTHOR]

ABSTRACT Bacterial species often consist of strains with variable gene content, collectively referred to as the pangenome. Variations in the genetic makeup of strains can alter bacterial physiology and fitness. To define biologically relevant genes of a genome, genome-wide transposon mutant libraries have been used to identify genes essential for survival or virulence in a given strain. Such phenotypic studies have been conducted in four different genotypes of the human pathogen Streptococcus pyogenes, yet challenges exist in comparing results across studies conducted in different genetic backgrounds and conditions. To advance genotype to phenotype inferences across different S. pyogenes strains, we built a pangenome database of 249 S. pyogenes reference genomes. We systematically re-analyzed publicly available transposon sequencing datasets from S. pyogenes using a transposon sequencing-specific analysis pipeline, Transit. Across four genetic backgrounds and nine phenotypic conditions, 355 genes were essential for survival, corresponding to ~24% of the core genome. Clusters of Orthologous Genes (COG) categories related to coenzyme and lipid transport and growth functions were overrepresented as essential. Finally, essential operons across S. pyogenes genotypes were defined, with an increased number of essential operons detected under in vivo conditions. This study provides an extendible database to which new studies can be added, and a searchable html-based resource to direct future investigations into S. pyogenes biology.IMPORTANCEStreptococcus pyogenes is a human-adapted pathogen occupying restricted ecological niches. Understanding the essentiality of genes across different strains and experimental conditions is important to direct research questions and efforts to prevent the large burden of disease caused by S. pyogenes. To this end we systematically reanalyzed transposon sequencing studies in S. pyogenes using transposon sequencing-specific methods, integrating them into an extendible meta-analysis framework. This provides a repository of gene essentiality in S. pyogenes which was used to highlight specific genes of interest and for the community to guide future phenotypic studies.

Flax is the main oil crop in China, and has been affected by flax wilt for a long time. The catalytic subunit of β-1, 3-glucan synthase (FKS/GLS) is necessary for cell wall synthesis, the biological function of which was analyzed in the study through cloning the gene, constructing FolGLS2 gene deletion mutant, observing phenotype and assaying the virulence of the gene disruption mutants. A full-length of 5947bp DNA sequence was obtained, which contained an open reading frame 5847bp, encoding a total of 1948 amino acids. Observation of the phenotype of the FolGLS2 gene deletion mutant showed that the colony of the mutant was small and compact, its growth rate decreased obviously, the mycelium was short, thick and no longer produced conidia. The cell wall of the mutant mycelium changed somehow, which was strongly resistant to the protoplasting buffer commonly used for wild type and was unable to release protoplasts. Infection assay to the flax seedling demonstrated that pathogenicity of deletion mutant was significantly decreased. Conclusively, FolGLS2 gene, which encodes catalytic subunit of β-1, 3- glucan synthase, plays a regulatory role in conidiogenesis, mycelial vegetative growth, cell wall properties and pathogenicity of Fusarium oxysporum f.sp.lini. [ABSTRACT FROM AUTHOR]

Prdx2 is a peroxiredoxin (Prx) family protein that protects cells from attack via reactive oxygen species (ROS), and it has an important role in improving the resistance and scavenging capacity of ROS in fungi. Arthrobotrys oligospora is a widespread nematode-trapping fungus that can produce three-dimensional nets to capture and kill nematodes. In this study, AoPrdx2, a homologous protein of Prx5, was investigated in A. oligospora via gene disruption, phenotypic analysis, and metabolomics. The deletion of Aoprdx2 resulted in an increase in the number of mycelial septa and a reduction in the number of nuclei and spore yield. Meanwhile, the absence of Aoprdx2 increased sensitivity to oxidative stresses, whereas the ∆Aoprdx2 mutant strain resulted in higher ROS levels than that of the wild-type (WT) strain. In particular, the inactivation of Aoprdx2 severely influenced trap formation and pathogenicity; the number of traps produced by the ∆Aoprdx2 mutant strain was remarkably reduced and the number of mycelial rings of traps in the ∆Aoprdx2 mutant strain was less than that of the WT strain. In addition, the abundance of metabolites in the ∆Aoprdx2 mutant strain was significantly downregulated compared with the WT strain. These results indicate that AoPrdx2 plays an indispensable role in the scavenging of ROS, trap morphogenesis, and secondary metabolism. [ABSTRACT FROM AUTHOR]

As industrial shochu yeast is a diploid strain, obtaining a strain with mutations in both allelic genes was considered difficult. We investigated a method for disrupting two copies of a homozygous gene with a single transformation. We designed a disruption cassette containing an intact LYS5 flanked by nonfunctional ura3 gene fragments divided into the 5′- and 3′-regions. These fragments had overlapping sequences that enabled LYS5 removal as well as URA3 regeneration through loop-out. Furthermore, both ends of the disruption cassette had an additional repeat sequence that allowed the cassette to be removed from the chromosome through loop-out. First, 45 bases of 5′- and 3′-regions of target gene sequences were added on both ends of this cassette using polymerase chain reaction; the resultant disruption cassette was introduced into a shochu yeast strain (ura3 / ura3 lys5 / lys5); then, single allele disrupted strains were selected on Lys drop-out plates; and after cultivation in YPD medium, double-disrupted strains, in which replacement of another allelic gene with disruption cassette by loss of heterozygosity and regeneration of URA3 in one of the cassettes by loop-out, were obtained by selection on Ura and Lys drop-out plates. The disruption cassettes were removed from the double-disrupted strain via loop-out between repeat sequences in the disruption cassette. The strains that lost either URA3 or LYS5 were counter-selected on 5-fluoroorotic acid or α-amino adipic acid plates, respectively. Using this method, we obtained leu2 / leu2 and leu2 / leu2 his3 / his3 strains in shochu yeast, demonstrating the effectiveness and repeatability of this gene disruption technique in diploid yeast Saccharomyces cerevisiae. [Display omitted] [ABSTRACT FROM AUTHOR]

ABSTRACT Pseudomonas syringae possesses genes for all five major DEAD-box RNA helicases, rhlE, srmB, csdA, dbpA, and rhlB, that are found in γ-proteobacteria. Bioinformatic analysis of the RNA helicase genes provided detailed insights into their genomic organization, promoter characteristics, regulatory 3′ regions, and sequence similarity. Disruption of rhlB and rhlE genes did not have any effect on growth of the P. syringae mutants at optimum (22°C) or at low (4°C) temperatures. On the other hand, disruption of srmB and dbpA genes caused a slow-growing phenotype in the mutants at low temperature (4°C). In comparison to the above-mentioned helicases, deletion of csdA gene caused the mutant (ΔcsdA) to be totally incapable of growing at 4°C while marginally affecting the growth (resulting in slow growth) at the optimum temperature (22°C). Functional complementation studies revealed that RNA helicases are functionally non-redundant, as the roles performed by different helicases are individual and specific. IMPORTANCE RNA metabolism is important as RNA acts as a link between genomic information and functional biomolecules, thereby playing a critical role in cellular response to environment. We investigated the role of DEAD-box RNA helicases in low-temperature adapted growth of P. syringae, as this group of enzymes play an essential role in modulation of RNA secondary structures. This is the first report on the assessment of all major DEAD-box RNA helicases in any Antarctic bacterium. Of the five RNA helicases, three (srmB, csdA, and dbpA) are important for the growth of the Antarctic P. syringae at low temperature. However, the requisite role of dbpA and the indispensable requirement of csdA for low-temperature adapted growth are a novel finding of this study. Growth analysis of combinatorial deletion strains was performed to understand the functional interaction among helicase genes. Similarly, genetic complementation of RNA helicase mutants was conducted for identification of gene redundancy in P. syringae.

Mead acid (MA; 20:3ω9) is one of the ω9 series of polyunsaturated fatty acids (PUFAs). MA is used to inhibit the inflammation of joints and is applied to the medicinal or health food field. We aimed to construct MA-producing strains with disruption of the Δ12-desaturase gene (Δ1 2ds) via an efficient gene-targeting system using the lig4 -disrupted strain of Mortierella alpina 1S-4 as the host. The transformants showed a unique fatty acid composition that only comprised ω9-PUFAs and saturated fatty acids, while ω6-and ω3-PUFAs were not detected, and the total composition of ω9-PUFAs, including oleic acid (18:1ω9), 18:2ω9, 20:1ω9, 20:2ω9, and MA, was up to 68.4% of the total fatty acids. The MA production in the Δ1 2ds -disruptant reached 0.10 g/L (8.5%), which exceeded 0.050 g/L (4.6%) in the conventional Δ1 2ds -defective mutant JT-180. [ABSTRACT FROM AUTHOR]

Three new cyclic lipopeptides, olenamidonins A-C (1–3), in addition to two previously reported metabolites (4 and 5), were accumulated in the ΔdtxRso deletion mutant of deepsea-derived Streptomyces olivaceus SCSIO 1071. The structures of these cyclic lipopeptides were determined by a combination of spectroscopic methods and circular dichroism (CD) measurement. The antibacterial assay results showed that compounds 1–5 displayed different degrees of growth inhibition against multidrug-resistant (MDR) bacterial strains Enterococcus faecalis CCARM 5172 and Enterococcus faecium CCARM 5203 with minimum inhibitory concentrations (MICs) of 1.56−6.25 μg/mL.

The transcription factor THCTF1 from Trichoderma harzianum, previously linked to the production of 6-pentyl-2H-pyran-2-one (6-PP) derivatives and antifungal activity against Fusarium oxysporum, has been related in this study to conidiation, production of an array of volatile organic compounds (VOCs) and expression of methyltransferase genes. VOCs emitted by three T. harzianum strains (wild type T34, transformant ΔD1-38 that is disrupted in the Thctf1 gene encoding the transcription factor THCTF1, and ectopic integration transformant ΔJ3-16) were characterized by Proton Transfer Reaction-Quadrupole interface-Time-Of-Flight-Mass Spectrometry (PTR-Qi-TOF-MS). Thctf1 disruption affected the production of numerous VOCs such as the antifungal volatiles 2-pentyl furan and benzaldehyde which were under-emitted, and acetoine, a plant systemic defense inductor, which was over-emitted. Biological assays show that VOCs regulated by THCTF1 are involved in the T. harzianum antifungal activity against Botrytis cinerea and in the beneficial effects leading to Arabidopsis plant development. The VOC blend from the disruptant ΔD1-38: (i) inhibited Arabidopsis seed germination for at least 26 days and (ii) when applied to Arabidopsis seedlings resulted in increased jasmonic acid- and salicylic acid-dependent defenses. [ABSTRACT FROM AUTHOR]

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Herein we describe, to our knowledge for the first time the use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 (CRISPR/Cas9) system for genome editing of Neospora caninum, an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide. By using plasmids containing the CRISPR/Cas9 components adapted to the closely related parasite Toxoplasma gondii, we successfully knocked out a green fluorescent protein (GFP) in an Nc-1 GFP-expressing strain, and efficiently disrupted the NcGRA7 gene in the Nc-Spain7 isolate by insertion of a pyrimethamine resistance cassette. The successful use of this technology in N. caninum lays the foundation for an efficient, targeted gene modification tool in this parasite.

Sea urchins have become significant mariculture species globally, and also serve as invertebrate model organisms in developmental biology. Cis-regulatory elements (enhancers) control development and physiology by regulating gene expression. Mutations that affect the function of these sequences may contribute to phenotypic diversity. Cis-regulatory targets offer new breeding potential for the future. Here, we use the CRISPR/Cas9 system to disrupt an enhancer of Endo16 in developing Lytechinus variegatus embryos, in consideration of the thorough research on Endo16's regulatory region. We designed six gRNAs against Endo16 Module A (the most proximal region of regulatory sequences, which activates transcription in the vegetal plate and archenteron, specifically) and discovered that Endo16 Module A-disrupted embryos failed to undergo gastrulation at 20 h post fertilization. This result partly phenocopies morpholino knockdowns of Endo16. Moreover, we conducted qPCR and clone sequencing experiments to verify these results. Although mutations were not found regularly from sequencing affected individuals, we discuss some potential causes. In conclusion, our study provides a feasible and informative method for studying the function of cis-regulatory elements in sea urchins, and contributes to echinoderm precision breeding technology innovation and aquaculture industry development. [ABSTRACT FROM AUTHOR]

Dothistroma needle blight, Cyclaneusma needle blight and red needle cast are devastating foliar pine diseases caused by the fungi Dothistroma septosporum and Cyclaneusma minus and the oomycete Phytophthora pluvialis, respectively. These pathogens colonise the host apoplast, secreting effector proteins to promote infection and disease. If these effectors are recognised by corresponding host resistance proteins, they activate the plant immune system to stop pathogen growth. We aimed to identify and characterise effectors that are common to all three pathogens. Using D. septosporum as a starting point, three candidate effectors (CEs) were investigated: Ds69335 (a CAP protein) and Ds131885, both of which have sequence and structural similarity to CEs of C. minus and P. pluvialis, and Ds74283, which adopts a β-trefoil fold and has structural rather than sequence similarity to CEs from all three pathogens. Notably, of the CEs investigated, Ds74283 and Ds131885, as well as their homologues from C. minus and P. pluvialis, elicited chlorosis or cell death in Nicotiana species, with Ds131885 and its homologues also triggering cell death in Pinus radiata. In line with these observed responses being related to activation of the plant immune system, the chlorosis triggered by Ds131885 and its homologues was compromised in a Nicotiana benthamiana mutant lacking the extracellular immune system co-receptor, SOBIR1. Such cross-kingdom, plant immune system-activating effectors, whether similar in sequence or structure, might ultimately enable the selection or engineering of durable, broad-spectrum resistance against foliar pine pathogens., (© 2025 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

CRISPR-Cas9 gene editing has become an important tool for the study of plant pathogens, allowing researchers to functionally characterize specific genes involved in phytopathogenicity, virulence, and fungicide resistance. Protocols for CRISPR-Cas9 gene editing have already been developed for Phytophthoras, an important group of oomycete plant pathogens; however, these efforts have exclusively focused on agricultural pathosystems, with research lacking for forest pathosystems. We sought to develop CRISPR-Cas9 gene editing in two forest pathogenic Phytophthoras, Phytophthora cactorum and P. ramorum , using a plasmid-ribonucleoprotein (RNP) co-transformation approach. Our gene target in both species was the ortholog of PcORP1 , which encodes an oxysterol-binding protein that is the target of the fungicide oxathiapiprolin in the agricultural pathogen P. capsici . We delivered liposome complexes, each containing plasmid DNA and CRISPR-Cas9 RNPs, to Phytophthora protoplasts using a polyethylene glycol-mediated transformation protocol. We obtained two ORP1 mutants in P. cactorum but were unable to obtain any mutants in P. ramorum . The two P. cactorum mutants exhibited decreased resistance to oxathiapiprolin, as measured by their radial growth relative to wild-type cultures on oxathiapiprolin-supplemented medium. Our results demonstrate the potential for RNP-mediated CRISPR-Cas9 gene editing in P. cactorum and provide a foundation for future optimization of our protocol in other forest pathogenic Phytophthora species.IMPORTANCECRISPR-Cas9 gene editing has become a valuable tool for characterizing the genetics driving virulence and pathogenicity in plant pathogens. CRISPR-Cas9 protocols are now well-established in several Phytophthora species, an oomycete genus with significant economic and ecological impact globally. These protocols, however, have been developed for agricultural Phytophthora pathogens only; CRISPR-Cas9 systems have not yet been developed for any forest pathogenic Phytophthoras. In this study, we sought to establish CRISPR-Cas9 gene editing in two forest Phytophthora pathogens that cause widespread tree mortality: P. cactorum and P. ramorum . We successfully obtained gene mutations in P. cactorum and demonstrated a decrease in fungicide resistance, a trait that could impact the pathogen's ability to cause disease. However, the same protocol did not yield any mutants in P. ramorum . The results of our study will serve as a baseline for the development of CRISPR-Cas9 gene editing in forest Phytophthoras and other oomycetes.

Abstract Nance–Horan syndrome (NHS) is a rare X-linked dominant disorder caused by mutation in the NHS gene on chromosome Xp22.13. (OMIM 302350). Classic NHS manifested in males is characterized by congenital cataracts, dental anomalies, dysmorphic facial features and occasionally intellectual disability. Females typically have a milder presentation. The majority of reported cases of NHS are the result of nonsense mutations and small deletions. Isolated X-linked congenital cataract is caused by non-recurrent rearrangement-associated aberrant NHS transcription. Classic NHS in females associated with gene disruption by balanced X-autosome translocation has been infrequently reported. We present a familial NHS associated with translocation t(X;19) (Xp22.13;q13.1). The proband, a 28-year-old female, presented with intellectual disability, dysmorphic features, short stature, primary amenorrhea, cleft palate, and horseshoe kidney, but no NHS phenotype. A karyotype and chromosome microarray analysis (CMA) revealed partial monosomy Xp/partial trisomy 19q with the breakpoint at Xp22.13 disrupting the NHS gene. Family history revealed congenital cataracts and glaucoma in the patient’s mother, and congenital cataracts in maternal half-sister and maternal grandmother. The same balanced translocation t(X;19) was subsequently identified in both the mother and maternal half-sister, and further clinical evaluation of the maternal half-sister made a diagnosis of NHS. This study describes the clinical implication of NHS gene disruption due to balanced X-autosome translocations as a unique mechanism causing Nance–Horan syndrome, refines dose effects of NHS on disease presentation and phenotype expressivity, and justifies consideration of karyotype and fluorescence in situ hybridization (FISH) analysis for female patients with familial NHS if single-gene analysis of NHS is negative.

The use of broad-spectrum antimycotic therapy, immunosuppressive therapy, and indwelling medical devices has contributed to the increased frequency of mucosal and systemic infections caused by Candida glabrata. A major concern for C. glabrata and other Candida spp. infections is the increase in drug resistance. To address these issues, additional molecular tools for the study of C. glabrata are needed. In this investigation, we developed an Agrobacterium tumefaciens transformation system for C. glabrata. A number of parameters were investigated to determine their effect on transformation frequency, and then an optimized protocol was developed. The optimal conditions for the transformation of C. glabrata were found to be an infection incubation temperature of 26 °C, 0.2 mM acetosyringone in both induction media and co-culture media, 0.7% agar concentration, and a multiplicity of infection of 50:1 A. tumefaciens to C. glabrata. Importantly, the frequency of multiple integrations was low (5%), demonstrating that A. tumefaciens generally integrates at single sites in C. glabrata, which is consistent with other fungal A. tumefaciens transformation systems. The development of this system in C. glabrata adds another tool for the molecular manipulation of this increasingly important fungal pathogen. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus expresses diverse proteins at different stages of growth. The immunodominant staphylococcal antigen A (IsaA) is one of the proteins that is constitutively produced by S. aureus during colonisation and infection. SACOL2584 (or isaA) is the gene that encodes this protein. It has been suggested that IsaA can hydrolyse cell walls, and there is still need to study isaA gene disruption to analyse its impact on staphylococcal phenotypes and on alteration to its transcription and protein profiles. In the present study, the growth curve in RPMI medium (which mimics human plasma), autolytic activity, cell wall morphology, fibronectin and fibrinogen adhesion and biofilm formation of S. aureus SH1000 (wildtype) was compared to that of S. aureus MS001 (isaA mutant). RNA sequencing and liquid chromatography–tandem mass spectrometry were carried out on samples of both S. aureus strains taken during the exponential growth phase, followed by bioinformatics analysis. Disruption of isaA had no obvious effect on the growth curve and autolysis ability or thickness of cell walls, but this study revealed significant strength of fibronectin adherence in S. aureus MS001. In particular, the isaA mutant formed less biofilm than S. aureus SH1000. In addition, proteomics and transcriptomics showed that the adhesin/biofilm-related genes and hemolysin genes, such as sasF, sarX and hlgC, were consistently downregulated with isaA gene disruption. The majority of the upregulated genes or proteins in S. aureus MS001 were pur genes. Taken together, this study provides insight into how isaA disruption changes the expression of other genes and has implications regarding biofilm formation and biological processes. [ABSTRACT FROM AUTHOR]

Dothistroma needle blight, caused by Dothistroma septosporum, has increased in incidence and severity over the last few decades and is now one of the most important global diseases of pines. Disease resistance breeding could be accelerated by knowledge of pathogen virulence factors and their host targets. However, this is hindered due to inefficient targeted gene disruption in D. septosporum, which is required for virulence gene characterisation. Here we report the first successful application of CRISPR/Cas9 gene editing to a Dothideomycete forest pathogen, D. septosporum. Disruption of the dothistromin pathway regulator gene AflR, with a known phenotype, was performed using nonhomologous end-joining repair with an efficiency of >90%. Transformants with a range of disruption mutations in AflR were produced. Disruption of Ds74283, a D. septosporum gene encoding a secreted cell death elicitor, was also achieved using CRISPR/Cas9, by using a specific donor DNA repair template to aid selection where the phenotype was unknown. In this case, 100% of screened transformants were identified as disruptants. In establishing CRISPR/Cas9 as a tool for gene editing in D. septosporum, our research could fast track the functional characterisation of candidate virulence factors in D. septosporum and helps set the foundation for development of this technology in other forest pathogens. [ABSTRACT FROM AUTHOR]

Structural variation is a major type of genetic variation that can potentially induce powerful genetic effects. In this study, we examined the Inv(A07)p1.09p2.23 genetic inversion in brown fibre cotton at the individual and population genetics levels. A dark-brown fibre mutant that resulted from a distant hybridization between Gossypium barbadense and G. hirsutum, and a natural population including 30 dark-brown, 70 light-brown and 21 white fibre cotton accessions were collected to perform a functional study of this micro-inversion. The results showed that Inv(A07)p1.09p2.23 can be detected by high-throughput resequencing method, and induce micro-deletion, gene disruption (Ghir_A07G000980) and abnormal gene expression in the breakpoint regions. Inv(A07)p1.09p2.23 existed in only dark-brown fibre cotton, had undergone negative selection in elite brown fibre cultivars, and was significantly associated with fibre colour and nine fibre traits. In the Inv(A07)p1.09p2.23 region, nucleotide diversity was lower, recombination was absent, and linkage disequilibrium was higher. Overall, this inversion event in dark-brown fibre cotton produced significant genetic effects, and this study will guide us to better understand the genetic effects of inversion events in dark-brown fibre cotton.

Control of both human and canine leishmaniasis is based on a very short list of chemotherapeutic agents, headed by antimonial derivatives (Sb). The utility of these molecules is severely threatened by high rates of drug resistance. The ABC transporter MRPA is one of the few key Sb resistance proteins described to date, whose role in detoxification has been thoroughly studied in Leishmania parasites. Nonetheless, its rapid amplification during drug selection complicates the discovery of other mechanisms potentially involved in Sb resistance. In this study, stepwise drug-resistance selection and next-generation sequencing were combined in the search for novel Sb-resistance mechanisms deployed by parasites when MRPA is abolished by targeted gene disruption. The gene mrpA is not essential in L. infantum, and its disruption leads to an Sb hypersensitive phenotype in both promastigotes and amastigotes. Five independent mrpA−/- mutants were selected for antimony resistance. These mutants displayed major changes in their ploidy, as well as extrachromosomal linear amplifications of the subtelomeric region of chromosome 23, which includes the genes coding for ABCC1 and ABCC2. Overexpression of ABCC2, but not of ABCC1, resulted in increased Sb tolerance in the mrpA−/- mutant. SNP analyses revealed three different heterozygous mutations in the gene coding for a serine acetyltransferase (SAT) involved in de novo cysteine synthesis in Leishmania. Overexpression of satQ390K, satG321R and satG325R variants led to a 2–3.2 -fold increase in Sb resistance in mrpA−/- parasites. Only satG321R and satG325R induced increased Sb resistance in wild-type parasites. These results reinforce and expand knowledge on the complex nature of Sb resistance in Leishmania parasites.

Abstract Background Gene disruption in mouse embryonic stem cells or zygotes is a conventional genetics approach to identify gene function in vivo. However, because different gene disruption strategies use different mechanisms to disrupt genes, the strategies can result in diverse phenotypes in the resulting mouse model. To determine whether different gene disruption strategies affect the phenotype of resulting mutant mice, we characterized Rhbdf1 mouse mutant strains generated by three commonly used strategies—definitive-null, targeted knockout (KO)-first, and CRISPR/Cas9. Results We find that Rhbdf1 responds differently to distinct KO strategies, for example, by skipping exons and reinitiating translation to potentially yield gain-of-function alleles rather than the expected null or severe hypomorphic alleles. Our analysis also revealed that at least 4% of mice generated using the KO-first strategy show conflicting phenotypes. Conclusions Exon skipping is a widespread phenomenon occurring across the genome. These findings have significant implications for the application of genome editing in both basic research and clinical practice.

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

To increase protein production, technologies of gene manipulation for engineering the yeast Komagataella phaffii are extensively exploited. In this study, we developed a convenient gene disruption method in the yeast via Cre/ loxP system. First, the simple gene disruption cassette [upstream homologous region (UP)- lox71 - Sh ble - lox66 -downstream homologous region (DW)] was constructed and transformed into the yeast to replace target gene. Second, the Sh ble gene of the cassette integrated in the chromosome was inserted with the auxiliary plasmid pPICZαA/cre/his4, resulting in an expanded cassette of UP- lox71 - Sh ble -pPICZαA/cre/his4- lox66 -DW. The auxiliary plasmid was generated via sequential insertion of cre and hi s 4 genes into pPICZαA, and linearized with Sma I before its transformation. Finally, for deletion of the sequence between lox71 and lox66 sites in the expanded cassette, CRE protein responsible for Cre/ loxP- mediated recombination was produced by methanol induction. Consequently, the corresponding sequence was eliminated permanently, only leaving a scar of lox72 site in the disrupted genes. This strategy was verified by disrupting two genes in the yeast. As the markers were recycled, it was also suitable for multiple gene disruption. • A simple structure of gene disruption cassette was applied. • A prefabricated sequence facilitated the construction of gene disruption cassette. • The auxiliary plasmid was ready-made for sequential deletion of multiple genes. • Efficient deletion of the targeted sequence between lox71 and lox66 was observed. [ABSTRACT FROM AUTHOR]

Nance–Horan syndrome (NHS) is a rare X-linked dominant disorder caused by mutation in the NHS gene on chromosome Xp22.13. (OMIM 302350). Classic NHS manifested in males is characterized by congenital cataracts, dental anomalies, dysmorphic facial features and occasionally intellectual disability. Females typically have a milder presentation. The majority of reported cases of NHS are the result of nonsense mutations and small deletions. Isolated X-linked congenital cataract is caused by non-recurrent rearrangement-associated aberrant NHS transcription. Classic NHS in females associated with gene disruption by balanced X-autosome translocation has been infrequently reported. We present a familial NHS associated with translocation t(X;19) (Xp22.13;q13.1). The proband, a 28-year-old female, presented with intellectual disability, dysmorphic features, short stature, primary amenorrhea, cleft palate, and horseshoe kidney, but no NHS phenotype. A karyotype and chromosome microarray analysis (CMA) revealed partial monosomy Xp/partial trisomy 19q with the breakpoint at Xp22.13 disrupting the NHS gene. Family history revealed congenital cataracts and glaucoma in the patient's mother, and congenital cataracts in maternal half-sister and maternal grandmother. The same balanced translocation t(X;19) was subsequently identified in both the mother and maternal half-sister, and further clinical evaluation of the maternal half-sister made a diagnosis of NHS. This study describes the clinical implication of NHS gene disruption due to balanced X-autosome translocations as a unique mechanism causing Nance–Horan syndrome, refines dose effects of NHS on disease presentation and phenotype expressivity, and justifies consideration of karyotype and fluorescence in situ hybridization (FISH) analysis for female patients with familial NHS if single-gene analysis of NHS is negative. [ABSTRACT FROM AUTHOR]

Abstract Background Botryosphaeria dothidea causes apple white rot and infects many tree plants. Genome data for B. dothidea are available and many pathogenesis-related genes have been predicted. However, a gene manipulation method is needed to study the pathogenic mechanism of B. dothidea. Results We established a gene disruption (GD) method based on gene homologous recombination (GHR) for B. dothidea using polyethylene glycol-mediated protoplast transformation. The results showed that a GHR cassette gave much higher GD efficiency than a GHR plasmid. A high GD efficiency (1.3 ± 0.14 per 106 protopasts) and low frequency of random insertions were achieved with a DNA cassette quantity of 15 μg per 106 protoplasts. Moreover, we successfully disrupted genes in two strains. Bdo_05381-disrupted transformants produced less melanin, whereas the Bdo_02540-disrupted transformant showed a slower growth rate and a stronger resistance to Congo red. Conclusion The established GD method is efficient and convenient and has potential for studying gene functions and the pathogenic mechanisms of B. dothidea and other coenocytic fungi.

ABSTRACT In order to provide new insights into the various activities of GH in specific tissues, recent advances have allowed for the generation of tissue-specific GHR knockout mice. To date, 21 distinct tissue-specific mouse lines have been created and reported in 28 publications. Targeted tissues include liver, muscle, fat, brain, bone, heart, intestine, macrophage, pancreatic beta cells, hematopoietic stem cells, and multi-tissue “global”. In this review, we provide a brief history and description of the 21 tissue-specific GHR knockout mouse lines. Arch Endocrinol Metab. 2019;63(6):557-67

3-(2-Hydroxyethyl)catechol (HEC) is a polyphenol reported to exhibit skin-lightning and antioxidative effects, and hence is expected to be used as cosmetic and food additives and chemical products such as electronic materials. In this study, we established biocatalytic HEC production from 2-phenylethanol using the dioxygenase whose expression was induced by toluene, CumA, and its flanking dehydrogenase, CumB, from an isolated strain, Pseudomonas sp. K17. Escherichia coli cells coexpressing CumA and CumB were stained blue during cultivation in Luria–Bertani medium, and HEC was not produced upon using the cell-free extracts as biocatalysts, likely resulting from the inhibitory effects of the blue dyes. The disruption of the tryptophanase gene of E. coli was found to repress the generation of the blue dyes, and enhanced HEC production. The blue dyes were extracted from the cell-free extracts, and their molecular formula was C 16 H 10 N 2 O 3 , suggesting they were monooxygenated indigo or its isomers. Although repression of blue dye formation and enhancement of HEC production were observed when cells were cultivated with glucose, the percent yield of HEC was 84% at 20 h, whereas that with tryptophanase disruption strain was 84% at 4 h. It was suggested that tryptophanase gene disruption could contribute to more efficient HEC production. [ABSTRACT FROM AUTHOR]

The N-end rule pathway is a regulated protein degradation system. Arthrobotrys oligospora, a typical nematode-trapping fungus, switches its life strategies from saprophytism to carnivorism when capturing free-living nematodes by means of adhesive networks. In this study, a putative E3-ligase AoUBR1 involved in N-end rule pathway was characterized in A. oligospora during vegetative growth and trap formation. Expression of AoUBR1 coding gene was down-regulated during trap formation. Compared with wild type, the AoUBR1 knock-out mutants decreased the vegetative growth, formed less traps, and turned to be sensitive to cold stress, while, AoUBR1 overexpression mutants lost the capacity to produce conidia and also formed less traps. A number of genes differentially expressed by knock-out and overexpression of AoUBR1 , which lead to the transcriptional responses associated with plasma membrane, transportation, oxidation, and proteolysis. AoUBR1 knock-out also resulted in the down-regulation of numerous secreted proteins associated with carnivorism and nutrient utilization from nematodes. In addition, AoUBR1 homologs were conserved in nematode-trapping fungi based on the genome searching. Therefore, the results suggested AoUBR1 in A. oligospora and its homologs in other trapping fungi are involved in the lifestyle switch between saprophytism and carnivorism. • AoUBR1 is a E3 ubiquitin-protein ligase in Arthrobotrys oligospora. • The lack of AoUBR1 impairs the vegetative growth in A. oligospora and affects the conidiation process. • AoUBR1 knock-out results in the decreased trap-formation and therefore reduces the virulence of A. oligospora. • AoUBR1 is necessary to the secretory enzymes, which are a digestive fluid directed against nematodes. [ABSTRACT FROM AUTHOR]

Abstract Background CRISPR/Cas9 has wide application potentials in a variety of biological species including Trichoderma reesei, a filamentous fungus workhorse for cellulase production. However, expression of Cas9 heterologously in the host cell could be time-consuming and sometimes even troublesome. Results We tested two gene disruption methods in T. reesei using CRISPR/Cas9 in this study. The intracellularly expressed Cas9 led to unexpected off-target gene disruption in T. reesei QM9414, favoring inserting 9- or 12-bp at 70- and 100-bp downstream of the targeted ura5. An alternative method was, therefore, established by assembling Cas9 and gRNA in vitro, followed by transformation of the ribonucleoprotein complex with a plasmid containing the pyr4 marker gene into T. reesei TU-6. When the gRNA targeting cbh1 was used, eight among the twenty seven transformants were found to lose the ability to express CBH1, indicative of successful cbh1 disruption through genome editing. Large DNA fragments including the co-transformed plasmid, chromosomal genes, or a mixture of these nucleotides, were inserted in the disrupted cbh1 locus. Conclusions Direct transformation of Cas9/gRNA complex into the cell is a fast means to disrupt a gene in T. reesei and may find wide applications in strain improvement and functional genomics study.

Staphylococcus aureus expresses diverse proteins at different stages of growth. The immunodominant staphylococcal antigen A (IsaA) is one of the proteins that is constitutively produced by S. aureus during colonisation and infection. SACOL2584 (or isaA) is the gene that encodes this protein. It has been suggested that IsaA can hydrolyse cell walls, and there is still need to study isaA gene disruption to analyse its impact on staphylococcal phenotypes and on alteration to its transcription and protein profiles. In the present study, the growth curve in RPMI medium (which mimics human plasma), autolytic activity, cell wall morphology, fibronectin and fibrinogen adhesion and biofilm formation of S. aureus SH1000 (wildtype) was compared to that of S. aureus MS001 (isaA mutant). RNA sequencing and liquid chromatography–tandem mass spectrometry were carried out on samples of both S. aureus strains taken during the exponential growth phase, followed by bioinformatics analysis. Disruption of isaA had no obvious effect on the growth curve and autolysis ability or thickness of cell walls, but this study revealed significant strength of fibronectin adherence in S. aureus MS001. In particular, the isaA mutant formed less biofilm than S. aureus SH1000. In addition, proteomics and transcriptomics showed that the adhesin/biofilm-related genes and hemolysin genes, such as sasF, sarX and hlgC, were consistently downregulated with isaA gene disruption. The majority of the upregulated genes or proteins in S. aureus MS001 were pur genes. Taken together, this study provides insight into how isaA disruption changes the expression of other genes and has implications regarding biofilm formation and biological processes.

Dothistroma needle blight, caused by Dothistroma septosporum, has increased in incidence and severity over the last few decades and is now one of the most important global diseases of pines. Disease resistance breeding could be accelerated by knowledge of pathogen virulence factors and their host targets. However, this is hindered due to inefficient targeted gene disruption in D. septosporum, which is required for virulence gene characterisation. Here we report the first successful application of CRISPR/Cas9 gene editing to a Dothideomycete forest pathogen, D. septosporum. Disruption of the dothistromin pathway regulator gene AflR, with a known phenotype, was performed using nonhomologous end-joining repair with an efficiency of >90%. Transformants with a range of disruption mutations in AflR were produced. Disruption of Ds74283, a D. septosporum gene encoding a secreted cell death elicitor, was also achieved using CRISPR/Cas9, by using a specific donor DNA repair template to aid selection where the phenotype was unknown. In this case, 100% of screened transformants were identified as disruptants. In establishing CRISPR/Cas9 as a tool for gene editing in D. septosporum, our research could fast track the functional characterisation of candidate virulence factors in D. septosporum and helps set the foundation for development of this technology in other forest pathogens.

To investigate the effect of fatty alcohol oxidase (FA0) gene disruption on Candida tropicalis, FA01 and FA02 genes were knocked out and complemented by homologous recombination.The growth of mutants and the changes of intracellular fatty alcohol oxidase activity were tested, and the ability of cells to synthesize fatty alcohols using alkanes was further evaluated. Strains FTYT(FA011/FA012), 2aT2bT (FA02a/FA026) and F3YT ( FA011/FA012AFA02a/FA026 ) were successfully constructed.All strains performed similar growth pattern to the control strain when using glucose as a carbon source.However, when using re-alkane( C12-C14) as carbon sources, the growth condition of each strain was different.The growth rate of FTYT strain was faster than other strains, and F3YT strain was significantly inhibited,indicating that the disruption of FA01 and FA02 genes impeded the ability of cells to utilize alkanes and affected cell growth. Compared with the parent strain, both the mutant strains of FTYT (F011 /F012) and F3YT (FA011 /FA012FA02a/FA02b) could convert alkane to 1-dodecanol and the latter strain could accumulate 59. 63 mg/L 1-dodecanol in shake flask fermentation level.FA01 and FA02 genes play an important role in the utilization of alkanes in cells and can serve as potential targets for the metabolic engineering of C.tropicalis for fatty alcohols production. [ABSTRACT FROM AUTHOR]

Inthomycins belong to growing family of oxazole-containing polyketides, and exhibit a broad spectrum of anti-oomycete and herbicidal activities. In this study, we purified inthomycins A and B from the metabolites of Streptomyces sp. SYP-A7193 and determined their chemical structures. Genome sequencing, comparative genomic analysis, and gene disruption of Streptomyces sp. SYP-A7193 showed that the inthomycin biosynthetic gene cluster (itm) belonged to the hybrid polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) system. Functional domain comparison and disruption/complementation experiments of itm12 resulted in the complete loss of inthomycins A and B and the subsequent restoration of their production, confirming that itm12 encodes a discrete acyltransferase and hence itm was considered to belong to the trans-AT type I PKS system. Moreover, the disruption/complementation experiments of itm15 also resulted in the loss and restoration of inthomycins A and B formation. Further gene cloning, expression, purification and activity verification of itm15 revealed that Itm15 as a cyclodehydratase that catalyzes a straight chain dehydration reaction to form an oxazole ring for the biosynthesis of inthomycins A and B. Thus, we discovered a novel enzyme that catalyzes oxazole ring formation and elucidated the complete biosynthetic pathway of inthomycins. [ABSTRACT FROM AUTHOR]

FR901533 (1, also known as WS79089B), WS79089A (2), and WS79089C (3) are polycyclic aromatic natural products with promising inhibitory activity to endothelin-converting enzymes. In this work, we isolated five tridecaketide products from Streptosporangium roseum No. 79089, including 1–3, benaphthamycin (4) and a novel FR901533 analogue (5). The structure of 5 was characterized based on spectroscopic data. Compared with the major product 2, the new compound 5 has an additional hydroxyl group at C-12 and an extra methyl group at the 13-OH. The configuration of C-19 of these compounds was determined to be R using Mosher's method. A putative biosynthetic gene cluster for compounds 1–5 was discovered by analyzing the genome of S. roseum No. 79089. This 38.6-kb gene cluster contains 38 open reading frames, including a minimal polyketide synthase (wsaA-C), an aromatase (wsaD), three cyclases (wsaE, F, and W), and a series of tailoring enzymes such as monooxygenases (wsaO1-O7) and methyltransferases (wsaM1 and M2). Disruption of the ketosynthase gene (wsaA) in this gene cluster abolished the production of 1–5, confirming that this gene cluster is indeed responsible for the biosynthesis of 1–5. A type II polyketide biosynthetic pathway was proposed for this group of natural endothelin-converting enzyme inhibitors. Key points: • Five aromatic tridecaketides were isolated from Streptosporangium roseum No. 79089. • A novel FR901533 analogue, 12-hydroxy-13-O-methyl-WS79089A, was characterized. • The absolute configuration of C-19 of FR901533 and analogues was determined. • The biosynthetic gene cluster of FR901533 and analogues was discovered. [ABSTRACT FROM AUTHOR]

Control of both human and canine leishmaniasis is based on a very short list of chemotherapeutic agents, headed by antimonial derivatives (Sb). The utility of these molecules is severely threatened by high rates of drug resistance. The ABC transporter MRPA is one of the few key Sb resistance proteins described to date, whose role in detoxification has been thoroughly studied in Leishmania parasites. Nonetheless, its rapid amplification during drug selection complicates the discovery of other mechanisms potentially involved in Sb resistance. In this study, stepwise drug-resistance selection and next-generation sequencing were combined in the search for novel Sb-resistance mechanisms deployed by parasites when MRPA is abolished by targeted gene disruption. The gene mrpA is not essential in L. infantum , and its disruption leads to an Sb hypersensitive phenotype in both promastigotes and amastigotes. Five independent mrpA −/- mutants were selected for antimony resistance. These mutants displayed major changes in their ploidy, as well as extrachromosomal linear amplifications of the subtelomeric region of chromosome 23, which includes the genes coding for ABCC1 and ABCC2. Overexpression of ABCC2, but not of ABCC1, resulted in increased Sb tolerance in the mrpA −/- mutant. SNP analyses revealed three different heterozygous mutations in the gene coding for a serine acetyltransferase (SAT) involved in de novo cysteine synthesis in Leishmania. Overexpression of sat Q390K , sat G321R and sat G325R variants led to a 2–3.2 -fold increase in Sb resistance in mrpA −/- parasites. Only sat G321R and sat G325R induced increased Sb resistance in wild-type parasites. These results reinforce and expand knowledge on the complex nature of Sb resistance in Leishmania parasites. Image 1 • mrpA is not an essential gene in Leishmania infantum parasites. • Disruption of mrpA leads to an antimony-hypersensitive phenotype in promastigotes and amastigotes. • L. infantum deploys secondary survival mechanisms to increase antimony tolerance in the absence of MRPA. • Overexpression of ABCC2 confers resistance to antimony in mrpA null mutants. • Different mutations in the sat gene confer resistance to antimony. [ABSTRACT FROM AUTHOR]

A novel gene (CAS2) specifically expressed during appressorium formation was isolated from Colletotrichum gloeosporioides using Differential Display RTPCR. CAS2 comprises 368 deduced amino acid residues and is 50% identical to a hypothetical protein from Chaetomium globosum. ProtFun 2.2 server analysis predicted that Cas2 functions as a transport and binding protein. Based on putative transmembrane domain prediction software (HMMTOP), Cas2 protein is composed of five alpha-helical transmembrane domains with a very short external N-terminus tail and long internal C-terminus. ExPASy ScanProsite analysis showed the presence of integrin beta chain cysteine-rich domain, N-myristoylation site, EGF-like domain, 2Fe-2S ferredoxins, iron-sulfur binding region, VWFC domain, fungal hydrophobins signature, membrane lipoprotein lipid attachment site, and Janus-faced atracotoxin (J-ACTX) family signature in CAS2 protein. Mutants with deleted CAS2 were not significantly different in terms of vegetative growth, conidiation, and appressoria production compared to wild type. However, the Cas2 mutant produced multipolar germination, a feature which distinguishes it from wild type strain. Interestingly, the mutant is non-virulent to mango fruits, indicating that CAS2 may encode proteins that function as novel virulence factors in fungal pathogens. [ABSTRACT FROM AUTHOR]

The Mycobacterium smegmatis genome contains many genes encoding putative drug efflux pumps. Yet with the exception of lfrA, it is not clear whether these genes contribute to the intrinsic drug resistance of this organism. We showed first by reverse transcription (RT)-PCR that several of these genes, including lfrA as well as the homologues of Mycobacterium tuberculosis Rv1145, Rv1146, Rv1877, Rv2846c (efpA) and Rv3065 (mmr and emrE), were expressed at detectable levels in the strain mc(2)155. Null mutants each carrying an in-frame deletion of these genes were then constructed in M. smegmatis. The deletions of the lfrA gene or mmr homologue rendered the mutant more susceptible to multiple drugs such as fluoroquinolones, ethidium bromide, and acriflavine (two- to eightfold decrease in MICs). The deletion of the efpA homologue also produced increased susceptibility to these agents but unexpectedly also resulted in decreased susceptibility to rifamycins, isoniazid, and chloramphenicol (two- to fourfold increase in MICs). Deletion of the Rv1877 homologue produced some increased susceptibility to ethidium bromide, acriflavine, and erythromycin. The upstream region of lfrA contained a gene encoding a putative TetR family transcriptional repressor, dubbed LfrR. The deletion of lfrR elevated the expression of lfrA and produced higher resistance to multiple drugs. Multidrug-resistant single-step mutants, independent of LfrA and attributed to a yet-unidentified drug efflux pump (here called LfrX), were selected in vitro and showed decreased accumulation of norfloxacin, ethidium bromide, and acriflavine in intact cells. Finally, use of isogenic beta-lactamase-deficient strains showed the contribution of LfrA and LfrX to resistance to certain beta-lactams in M. smegmatis.

Abstract Background CRISPR/Cas9 system is a powerful method to investigate the role of genes by introducing a mutation selectively and efficiently to specific genome positions in cell and animal lines. However, in primary neuron cultures, this method is affected by the issue that the effectiveness of CRISPR/Cas9 is different in each neuron. Here, we report an easy, quick and reliable method to identify mutants induced by the CRISPR/Cas9 system at a single neuron level, using immunocytochemistry (ICC) and fluorescence imaging. Results Dissociated cortical cells were transfected with CRISPR/Cas9 plasmids targeting the transcription factor cAMP-response element binding protein (CREB). Fluorescence ICC with CREB antibody and quantitative analysis of fluorescence intensity demonstrated that CREB expression disappeared in a fraction of the transfected neurons. The downstream FOS expression was also decreased in accordance with suppressed CREB expression. Moreover, dendritic arborization was decreased in the transfected neurons which lacked CREB immunoreactivity. Conclusions Detection of protein expression is efficient to identify individual postmitotic neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures. The present method composed of CRISPR/Cas9 system, ICC and fluorescence imaging is applicable to study the function of various genes at a single-neuron level.

We have previously shown deep-sea-derived Streptomyces koyangensis SCSIO 5802 to produce two types of active secondary metabolites, abyssomicins and candicidins. Here, we report the complete genome sequence of S. koyangensis SCSIO 5802 employing bioinformatics to highlight its potential to produce at least 21 categories of natural products. In order to mine novel natural products, the production of two polycyclic tetramate macrolactams (PTMs), the known 10-epi-HSAF (1) and a new compound, koyanamide A (2), was stimulated via inactivation of the abyssomicin and candicidin biosynthetic machineries. Detailed bioinformatics analyses revealed a PKS/NRPS gene cluster, containing 6 open reading frames (ORFs) and spanning ~16 kb of contiguous genomic DNA, as the putative PTM biosynthetic gene cluster (BGC) (termed herein sko). We furthermore demonstrate, via gene disruption experiments, that the sko cluster encodes the biosynthesis of 10-epi-HSAF and koyanamide A. Finally, we propose a plausible biosynthetic pathway to 10-epi-HSAF and koyanamide A. In total, this study demonstrates an effective approach to cryptic BGC activation enabling the discovery of new bioactive metabolites; genome mining and metabolic profiling methods play key roles in this strategy.