Targeted Gene Replacement Research Articles

Translocon Subunits of the COP9 Signalosome Complex Are a Central Hub for Regulating Multiple Photoresponsive Processes and Autophagic Flux in Magnaporthe oryzae.

Photodependent processes, including circadian rhythm, autophagy, ubiquitination, neddylation/deneddylation, and metabolite biosynthesis, profoundly influence microbial pathogenesis. Although a photomorphogenesis signalosome (COP9/CSN) has been identified, the mechanism by which this large complex contributes to the pathophysiological processes in filamentous fungi remains unclear. Here, we identified eight CSN complex subunits in the rice blast fungus Magnaporthe oryzae and functionally characterized the translocon subunits containing a nuclear export or localization signal (NES/NLS). Targeted gene replacement of these CSN subunits, including MoCSN3, MoCSN5, MoCSN6, MoCSN7, and MoCSN12, attenuated vegetative growth and conidiation and rendered the deletion strains nonpathogenic. MoCSN7 deletion significantly suppressed arachidonic acid catabolism, and compromised cell wall integrity in M. oryzae. Surprisingly, we also discovered that MoCSN subunits, particularly MoCsn7, are required for the cAMP-dependent regulation of autophagic flux. Therefore, MoCSN significantly contributes to morphological, physiological, and pathogenic differentiation in M. oryzae by fostering cross-talk between multiple pathways.

Heterologous Expression of Human IFNγ and Anti-IL17 Antibody in Leishmania tarentolae Promastigote.

Leishmania is an intracellular flagellate protozoan parasite that causes a wide range of clinical diseases in humans. The basis of immunological resistance against leishmaniasis depends on Thl reactions and is within the time period of cytokine function. In this study, human anti-IL17 antibody and IFNγ-producing promastigote were produced to be used in leishmanization. A sequence of light and heavy chains' gene of anti-IL17 antibody and human IFNγ (hIFNγ) was obtained from the NCBI database and synthesized in the ECORV reaction site in the plasmid pGH, which it's called pGH-hIFNγ-antiIL17. The synthesized part using the restriction enzyme ECORV was extracted from the plasmid and after purification by electroporation was transferred to Iranian lizard Leishmania (I.L.L). Evaluation of structural presence in the I.L.L genome at the level of DNA and mRNA was assessed. The expressions of hIFNγ and anti-IL17 were evaluated and confirmed using ELISA and western blot analysis. The hIFNγ secreted from the culture medium was collected at high concentrations of 124.36 ± 6.47pg/mL. Targeted gene replacement into the I.L.L genome was successfully performed for the first time using the pGH-hIFNγ-antiIL17 plasmid in an identical replacement process. Stabilized recombinant DNA contains a target gene that has no toxicity to the parasite. The effective achievement of producing a recombinant gene was done for the first time by replacing the I.L.L-CPC gene with plasmid pGH-hIFNγ-antiIL17 by targeted gene replacement. This cab can regulate the production of hIFNγ and anti-IL17. This makes it a viable choice for eliminating leishmania.

Gene duplication and deletion caused by over-replication at a fork barrier

Replication fork stalling can provoke fork reversal to form a four-way DNA junction. This remodelling of the replication fork can facilitate repair, aid bypass of DNA lesions, and enable replication restart, but may also pose a risk of over-replication during fork convergence. We show that replication fork stalling at a site-specific barrier in fission yeast can induce gene duplication-deletion rearrangements that are independent of replication restart-associated template switching and Rad51-dependent multi-invasion. Instead, they resemble targeted gene replacements (TGRs), requiring the DNA annealing activity of Rad52, the 3’-flap nuclease Rad16-Swi10, and mismatch repair protein Msh2. We propose that excess DNA, generated during the merging of a canonical fork with a reversed fork, can be liberated by a nuclease and integrated at an ectopic site via a TGR-like mechanism. This highlights how over-replication at replication termination sites can threaten genome stability in eukaryotes.

High-fidelity large-diversity monoclonal mammalian cell libraries by cell cycle arrested recombinase-mediated cassette exchange.

Mammalian cells carrying defined genetic variations have shown great potentials in both fundamental research and therapeutic development. However, their full use was limited by lack of a robust method to construct large monoclonal high-quality combinatorial libraries. This study developed cell cycle arrested recombinase-mediated cassette exchange (aRMCE), able to provide monoclonality, precise genomic integration and uniform transgene expression. Via optimized nocodazole-mediated mitotic arrest, 20% target gene replacement efficiency was achieved without antibiotic selection, and the improved aRMCE efficiency was applicable to a variety of tested cell clones, transgene targets and transfection methods. As a demonstration of this versatile method, we performed directed evolution of fragment crystallizable (Fc), for which error-prone libraries of over 107 variants were constructed and displayed as IgG on surface of CHO cells. Diversities of constructed libraries were validated by deep sequencing, and panels of novel Fc mutants were identified showing improved binding towards specific Fc gamma receptors and enhanced effector functions. Due to its large cargo capacity and compatibility with different mutagenesis approaches, we expect this mammalian cell platform technology has broad applications for directed evolution, multiplex genetic assays, cell line development and stem cell engineering.

Characterization of the RAD52 Gene in the Budding Yeast Naumovozyma castellii.

Several sources of DNA damage compromise the integrity and stability of the genome of every organism. Specifically, DNA double-strand breaks (DSBs) can have lethal consequences for the cell. To repair this type of DNA damage, the cells employ homology-directed repair pathways or non-homologous end joining. Homology-directed repair requires the activity of the RAD52 epistasis group of genes. Rad52 is the main recombination protein in the budding yeast Saccharomyces cerevisiae, and rad52Δ mutants have been characterized to show severe defects in DSB repair and other recombination events. Here, we identified the RAD52 gene in the budding yeast Naumovozyma castellii. Our analysis showed that the primary amino acid sequence of N. castellii Rad52 shared 70% similarity with S. cerevisiae Rad52. To characterize the gene function, we developed rad52Δ mutant strains by targeted gene replacement transformation. We found that N. castellii rad52Δ mutants showed lowered growth capacity, a moderately altered cell morphology and increased sensitivity to genotoxic agents. The decreased viability of the N. castellii rad52Δ mutants in the presence of genotoxic agents indicates that the role of the Rad52 protein in the repair of DNA damage is conserved in this species.

MoJMJD6, a Nuclear Protein, Regulates Conidial Germination and Appressorium Formation at the Early Stage of Pathogenesis in Magnaporthe oryzae.

In plant-pathogen interactions, Magnaporthe oryzae causes blast disease on more than 50 species of 14 monocot plants, including important crops such as rice, millet, and most 15 recently wheat. M. oryzae is a model fungus for studying plant-microbe interaction, and the main source for fungal pathogenesis in the field. Here we report that MoJMJD6 is required for conidium germination and appressorium formation in M. oryzae. We obtained MoJMJD6 mutants (ΔMojmjd6) using a target gene replacement strategy. The MoJMD6 deletion mutants were delayed for conidium germination, glycogen, and lipid droplets utilization and consequently had decreased virulence. In the ΔMojmjd6 null mutants, global histone methyltransferase modifications (H3K4me3, H3K9me3, H3K27me3, and H3K36me2/3) of the genome were unaffected. Taken together, our results indicated that MoJMJD6 function as a nuclear protein which plays an important role in conidium germination and appressorium formation in the M. oryzae. Our work provides insights into MoJMJD6-mediated regulation in the early stage of pathogenesis in plant fungi.

A CRISPR/Cas9-based multicopy integration system for protein production in Aspergillus niger.

The filamentous fungus Aspergillus niger is well known for its high protein secretion capacity and a preferred host for homologous and heterologous protein production. To improve the protein production capacity of A. niger even further, a set of dedicated protein production strains was made containing up to 10 glucoamylase landing sites (GLSs) at predetermined sites in the genome. These GLSs replace genes encoding enzymes abundantly present or encoding unwanted functions. Each GLS contains the promotor and terminator region of the glucoamylase gene (glaA), one of the highest expressed genes in A. niger. Integrating multiple gene copies, often realized by random integration, is known to boost protein production yields. In our approach the GLSs allow for rapid targeted gene replacement using CRISPR/Cas9-mediated genome editing. By introducing the same or different unique DNA sequences (dubbed KORE sequences) in each GLS and designing Cas9-compatible single guide RNAs, one is able to select at which GLS integration of a target gene occurs. In this way a set of identical strains with different copy numbers of the gene of interest can be easily and rapidly made to compare protein production levels. As an illustration of its potential, we successfully used the expression platform to generate multicopy A. niger strains producing the Penicillium expansum PatE::6xHis protein catalysing the final step in patulin biosynthesis. The A. niger strain expressing 10 copies of the patE::6xHis expression cassette produced about70 μg·mL-1 PatE protein in the culture medium with a purity just under 90%.

MRNA Vaccine Platform: mRNA Production and Delivery.

Vaccination is the most efficient way to prevent infectious diseases. mRNA-based vaccines is a new approach to vaccine development, which have several very useful advantages over other types of vaccines. Since mRNA encodes only the target antigen there is no potential risk of infection as in the case with attenuated or inactivated pathogens. The mode of action of mRNA-vaccines implies that their genetic information is expressed only in the cytosol, leaving very little possibility of mRNA integration into the host's genome. mRNA-vaccines can induce specific cellular and humoral immune responses, but do not induce the antivector immune response. The mRNA-vaccine platform allows for easy target gene replacement without the need to change the production technology, which is important to address the time lag between the epidemic onset and vaccine release. The present review discusses the history of mRNA vaccines, mRNA vaccine production technology, ways to increase mRNA stability, modifications of the cap, poly(A)-tail, coding and noncoding parts of mRNA, target mRNA vaccine purification from byproducts, and delivery methods.

CCR5 deficiency normalizes TIMP levels, working memory, and gamma oscillation power in APOE4 targeted replacement mice

The APOE4 allele increases the risk for Alzheimer's disease (AD) in a dose-dependent manner and is also associated with cognitive decline in non-demented elderly controls. In mice with targeted gene replacement (TR) of murine APOE with human APOE3 or APOE4, the latter show reduced neuronal dendritic complexity and impaired learning. APOE4 TR mice also show reduced gamma oscillation power, a neuronal population activity which is important to learning and memory. Published work has shown that brain extracellular matrix (ECM) can reduce neuroplasticity as well as gamma power, while attenuation of ECM can instead enhance this endpoint. In the present study we examine human cerebrospinal fluid (CSF) samples from APOE3 and APOE4 individuals and brain lysates from APOE3 and APOE4 TR mice for levels of ECM effectors that can increase matrix deposition and restrict neuroplasticity. We find that CCL5, a molecule linked to ECM deposition in liver and kidney, is increased in CSF samples from APOE4 individuals. Levels of tissue inhibitor of metalloproteinases (TIMPs), which inhibit the activity of ECM-degrading enzymes, are also increased in APOE4 CSF as well as astrocyte supernatants brain lysates from APOE4 TR mice. Importantly, as compared to APOE4/wild-type heterozygotes, APOE4/CCR5 knockout heterozygotes show reduced TIMP levels and enhanced EEG gamma power. The latter also show improved learning and memory, suggesting that the CCR5/CCL5 axis could represent a therapeutic target for APOE4 individuals.

In vitro</em></strong><strong> direct regeneration and <em>Agrobacterium tumefaciens</em> mediated <em>in planta</em> transformation of <em>Ocimum sanctum L.

An in vitro regeneration system for propagation has been successfully developed for a valuable medicinal and aromatic plant ‘ Ocimum sanctum L’. In the present study, petiole explants, from in-vitro grown cultures of O. sanctum, was used for direct regeneration. The developed protocol employed 98% of regeneration frequency in addition to 9.6 shoots per explant when cultured on Murashige and Skoog (MS) medium fortified with 3 mg/L benzylamino purine (BAP) and 1 mg/L Naphthalene acetic acid (NAA). Furthermore, Agrobacterium tumefaciens Mediated genetic Transformation (ATMT) protocol (transient and stable) was also developed using LBA4404 strain harboring pBI121 with uid-A and neomycin phosphotransferase genes. The regenerated transformants were shifted on MS with kanamycin (50 mg/L) and afterwards placed on the half-strength MS medium. The validation was done through polymerase chain reaction (PCR) with neomycin phosphotransferase-II ( npt-II) & β-glucoronidase ( uid-A) gene primers. The maximum stable transformation frequency of 70% ± 0.35 was achieved. Hence, it is apparent that the established protocols i.e. in vitro direct regeneration and ATMT are appropriate for integrating novel enzymes/genes through high throughput techniques such as gene tagging, and targeted gene replacement to modulate the primary as well as secondary metabolic flux towards desired agronomic product or trait in planta.

S1.3a Genetics andgenomics of Malassezia species

S1.3 Malassezia: genetics, genomics, and biology, September 21, 2022, 11:00 AM - 12:30 PM Malassezia includes yeasts belonging to the subphylum Ustilaginomycotina within the Basidiomycota. Malassezia yeasts are attracting the interest of both basic and applied scientists for their unique biological features, and for their importance in clinical and cosmetic settings. Although Malassezia yeasts are commonly found as commensal on human and animal skin, they are also associated with several skin disorders, such as dandruff/seborrheic dermatitis, atopic eczema, pityriasis versicolor, and folliculitis. More recently, an association of Malassezia with Crohn's disease, pancreatic ductal adenocarcinoma, and psoriasis exacerbation has been reported. To understand the genetic basis of Malassezia commensalism and pathogenicity, the availability of genomic and molecular tools plays a crucial role. Genomics advances in Malassezia reveal karyotype variations and gene turnover events, including genes horizontally transferred from bacteria. Moreover, the increasing availability of transcriptomic data allows us to prioritize studies on novel key genes that potentially characterize the pathophysiology of Malassezia fungi. For gene function studies, protocols for Agrobacterium tumefaciens-mediated transformation were developed and utilized in strategies of random insertional mutagenesis or targeted gene replacement through CRISPR/Cas9. Developed tools can be combined with the use of host-pathogen interaction models, such as the easy-to-use wax moth larvae of Galleria mellonella or the more complex murine skin model, enabling the characterization of both the fungal components that trigger skin damage and inflammation, and the inflammatory and antifungal response of the host to prevent fungal infection through immunological and molecular analyses of experimentally infected tissue.

Stemphylium lycopersici Nep1-like Protein (NLP) Is a Key Virulence Factor in Tomato Gray Leaf Spot Disease.

The fungus Stemphylium lycopersici (S. lycopersici) is an economically important plant pathogen that causes grey leaf spot disease in tomato. However, functional genomic studies in S. lycopersici are lacking, and the factors influencing its pathogenicity remain largely unknown. Here, we present the first example of genetic transformation and targeted gene replacement in S. lycopersici. We functionally analyzed the NLP gene, which encodes a necrosis- and ethylene-inducing peptide 1 (Nep1)-like protein (NLP). We found that targeted disruption of the NLP gene in S. lycopersici significantly compromised its virulence on tomato. Moreover, our data suggest that NLP affects S. lycopersici conidiospore production and weakly affects its adaptation to osmotic and oxidative stress. Interestingly, we found that NLP suppressed the production of reactive oxygen species (ROS) in tomato leaves during S. lycopersici infection. Further, expressing the fungal NLP in tomato resulted in constitutive transcription of immune-responsive genes and inhibited plant growth. Through gene manipulation, we demonstrated the function of NLP in S. lycopersici virulence and development. Our work provides a paradigm for functional genomics studies in a non-model fungal pathogen system.

Targeted Gene Knockouts by Protoplast Transformation in the Moss Physcomitrella patens.

Targeted gene knockout is particularly useful for analyzing gene functions in plant growth, signaling, and development. By transforming knockout cassettes consisting of homologous sequences of the target gene into protoplasts, the classical gene targeting method aims to obtain targeted gene replacement, allowing for the characterization of gene functions in vivo. The moss Physcomitrella patens is a known model organism for a high frequency of homologous recombination and thus harbors a remarkable rate of gene targeting. Other moss features, including easy to culture, dominant haploidy phase, and sequenced genome, make gene targeting prevalent in Physcomitrella patens. However, even gene targeting was powerful to generate knockouts, researchers using this method still experienced technical challenges. For example, obtaining a good number of targeted knockouts after protoplast transformation and regeneration disturbed the users. Off-target mutations such as illegitimate random integration mediated by nonhomologous end joining and targeted insertion wherein one junction on-target but the other end off-target is commonly present in the knockouts. Protoplast fusion during transformation and regeneration was also a problem. This review will discuss the advantages and technical challenges of gene targeting. Recently, CRISPR-Cas9 is a revolutionary technology and becoming a hot topic in plant gene editing. In the second part of this review, CRISPR-Cas9 technology will be focused on and compared to gene targeting regarding the practical use in Physcomitrella patens. This review presents an updated perspective of the gene targeting and CRISPR-Cas9 techniques to plant biologists who may consider studying gene functions in the model organism Physcomitrella patens.

Targeted Gene Insertion and Replacement in the Basidiomycete Ganoderma lucidum by Inactivation of Nonhomologous End Joining Using CRISPR/Cas9.

Targeted gene insertion or replacement is a promising genome-editing tool for molecular breeding and gene engineering. Although CRISPR/Cas9 works well for gene disruption and deletion in Ganoderma lucidum, targeted gene insertion and replacement remain a serious challenge due to the low efficiency of homologous recombination (HR) in this species. In this work, we demonstrate that the DNA double-strand breaks induced by Cas9 were mainly repaired via the nonhomologous end joining (NHEJ) pathway, at a frequency of 96.7%. To establish an efficient target gene insertion and replacement tool in Ganoderma, we first inactivated the NHEJ pathway via disruption of the Ku70 gene (ku70) using a dual single guide RNA (sgRNA)-directed gene deletion method. Disruption of the ku70 gene significantly decreased NHEJ activity in G. lucidum. Moreover, ku70 disruption strains exhibited 96.3% and 93.1% frequencies of targeted gene insertion and replacement, respectively, when target DNA with the orotidine 5'-monophosphate decarboxylase (ura3) gene and 1.5-kb homologous 5'- and 3'-flanking sequences was used as a donor template, compared to 3.3% and 0%, respectively, at these targeted sites for a control strain (Cas9 strain). Our results indicated that ku70 disruption strains were efficient recipients for targeted gene insertion and replacement. This tool will advance our understanding of functional genomics in G. lucidum. IMPORTANCE Functional genomic studies in Ganoderma have been hindered by the absence of adequate genome-engineering tools. Although CRISPR/Cas9 works well for gene disruption and deletion in G. lucidum, targeted gene insertion and replacement have remained a serious challenge due to the low efficiency of HR in these species, although such precise genome modifications, including site mutations, site-specific integrations, and allele or promoter replacements, would be incredibly valuable. In this work, we inactivated the NHEJ repair mechanism in G. lucidum by disrupting the ku70 gene using the CRISPR/Cas9 system. Moreover, we established a target gene insertion and replacement method in ku70-disrupted G. lucidum that possessed high-efficiency gene targeting. This technology will advance our understanding of the functional genomics of G. lucidum.

A reassessment of flocculosin-mediated biocontrol activity of Pseudozyma flocculosa through CRISPR/Cas9 gene editing

Pseudozyma flocculosa is an epiphytic yeast with powerful antagonistic activity against powdery mildews. This activity has been associated with the production of a rare antifungal glycolipid, flocculosin. In spite of the discovery of a specific gene cluster for flocculosin synthesis, attempts to ascribe a functional role to the molecule have been hampered by the inability to efficiently transform P. flocculosa. In this study, two different approaches, target gene replacement by homologous recombination (HR) and CRISPR-Cas9 based genome-editing, were utilized to decipher the role of flocculosin in the biocontrol activity of P.flocculosa. It was possible to alter the production of flocculosin through edition of fat1 by HR, but such mutants displayed abnormal phenotypes and the inability to produce sporidia. Sequencing analyses revealed that transformation by HR led to multiple insertions in the genome explaining the pleiotrophic effects of the approach. On the other hand, CRISPR-Cas9 transformation yielded one mutant that was altered specifically in the proper synthesis of flocculosin. Notwithstanding the loss of flocculosin production, such mutant was phenotypically similar to the wild-type, and when tested for its biocontrol activity against powdery mildew, displayed the same efficacy. These results offer strong evidence that flocculosin-mediated antibiosis is not responsible for the mode of action of P. flocculosa and highlight the potential of CRISPR-Cas9 for functional studies of otherwise difficult-to-transform fungi such as P. flocculosa.

Functional Analysis of Autophagy-Related Gene ATG12 in Potato Dry Rot Fungus Fusarium oxysporum.

Autophagy is an intracellular process in all eukaryotes which is responsible for the degradation of cytoplasmic constituents, recycling of organelles, and recycling of proteins. It is an important cellular process responsible for the effective virulence of several pathogenic plant fungal strains, having critical impacts on important crop plants including potatoes. However, the detailed physiological mechanisms of autophagy involved in the infection biology of soil-borne pathogens in the potato crop needs to be investigated further. In this study, the autophagy-related gene, FoATG12, in potato dry rot fungus Fusarium oxysporum was investigated by means of target gene replacement and overexpression. The deletion mutant ∆FoATG12 showed reduction in conidial formation and exhibited impaired aerial hyphae. The FoATG12 affected the expression of genes involved in pathogenicity and vegetative growth, as well as on morphology features of the colony under stressors. It was found that the disease symptoms were delayed upon being inoculated by the deletion mutant of FoATG12 compared to the wild-type (WT) and overexpression (OE), while the deletion mutant showed the disease symptoms on tomato plants. The results confirmed the significant role of the autophagy-related ATG12 gene in the production of aerial hyphae and the effective virulence of F. oxysporum in the potato crop. The current findings provid an enhanced gene-level understanding of the autophagy-related virulence of F. oxysporum, which could be helpful in pathogen control research and could have vital impacts on the potato crop.

SEC6 exocyst subunit contributes to multiple steps of growth and development of Physcomitrella (Physcomitrium patens).

Spatially directed cell division and expansion is important for plant growth and morphogenesis and relies on cooperation between the cytoskeleton and the secretory pathway. The phylogenetically conserved octameric complex exocyst mediates exocytotic vesicle tethering at the plasma membrane. Unlike other exocyst subunits of land plants, the core exocyst subunit SEC6 exists as a single paralog in Physcomitrium patens and Arabidopsis thaliana genomes. Arabidopsis SEC6 (AtSEC6) loss-of-function (LOF) mutation causes male gametophytic lethality. Our attempts to inactivate the P.patens SEC6 gene, PpSEC6, using targeted gene replacement produced two independent partial LOF ('weak allele') mutants via perturbation of the PpSEC6 gene locus. These mutants exhibited the same pleiotropic developmental defects: protonema with dominant chloronema stage; diminished caulonemal filament elongation rate; and failure in post-initiation gametophore development. Mutant gametophore buds, mostly initiated from chloronema cells, exhibited disordered cell file organization and cross-wall perforations, resulting in arrested development at the eight- to 10-cell stage. Complementation of both sec6 moss mutant lines by both PpSEC6 and AtSEC6 cDNA rescued gametophore development, including sexual organ differentiation. However, regular sporophyte formation and viable spore production were recovered only by the expression of PpSEC6, whereas the AtSEC6 complementants were only rarely fertile, indicating moss-specific SEC6 functions.

The GTPase-Activating Protein FgGyp1 Is Important for Vegetative Growth, Conidiation, and Virulence and Negatively Regulates DON Biosynthesis in Fusarium graminearium.

Ypt1 is a small Rab GTPase in yeast, Gyp1 functions at the Golgi as a negative regulator of Ypt1. Gyp1 homologs are conserved in filamentous fungi. However, the roles of Gyp1 in phytopathogenic fungi are still unclear. Herein, we investigated the functions of FgGyp1 in the wheat pathogen Fusarium graminearum by live-cell imaging, genetic, and pathological analyses. Targeted gene replacement method was used to delete FgGYP1 in F. graminearum. Phenotypic analyses showed that FgGyp1 is critically important not only for the vegetative growth of F. graminearum but also its conidiation. The mutant’s vegetative growth was significantly reduced by 70% compared to the wild type PH-1. The virulence of FgGYP1 deletion mutant was significantly decreased when compared with the wild type PH-1. We further found that FgGyp1 negatively regulates DON production of the fungus. Live-cell imaging clearly demonstrated that FgGyp1 mainly localizes to the Golgi apparatus. Moreover, the TBC domain, C-terminal, and N-terminal regions of FgGyp1 are found to be indispensable for its biological functions and normal localization. The Arg357 residue of FgGyp1 is essential for its functions but dispensable for the normal localization of the protein, while the Arg284 residue is not required for both the functions and normal localization of the protein. Furthermore, we showed that FgGyp1 essentially hydrolyzes the GTP-bound FgRab1 (activated form) to its corresponding GDP-bound (inactive) form in vitro, suggesting that FgGyp1 is a GTPase-activating protein (GAP) for FgRab1. Finally, FgGyp1 was found to be important for FgSnc1-mediated fusion of secretory vesicles from the Golgi with the plasma membrane in F. graminearum. Put together, these data demonstrate that FgGyp1 functions as a GAP for FgRab1 and is important for vegetative growth, conidiation and virulence, and negatively regulates DON biosynthesis in F. graminearum.

Potential Therapeutic Targets for Olfactory Dysfunction in Ciliopathies Beyond Single-Gene Replacement.

Olfactory dysfunction is a common disorder in the general population. There are multiple causes, one of which being ciliopathies, an emerging class of human hereditary genetic disorders characterized by multiple symptoms due to defects in ciliary biogenesis, maintenance, and/or function. Mutations/deletions in a wide spectrum of ciliary genes have been identified to cause ciliopathies. Currently, besides symptomatic therapy, there is no available therapeutic treatment option for olfactory dysfunction caused by ciliopathies. Multiple studies have demonstrated that targeted gene replacement can restore the morphology and function of olfactory cilia in olfactory sensory neurons and further re-establish the odor-guided behaviors in animals. Therefore, targeted gene replacement could be potentially used to treat olfactory dysfunction in ciliopathies. However, due to the potential limitations of single-gene therapy for polygenic mutation-induced diseases, alternative therapeutic targets for broader curative measures need to be developed for olfactory dysfunction, and also for other symptoms in ciliopathies. Here we review the current understanding of ciliogenesis and maintenance of olfactory cilia. Furthermore, we emphasize signaling mechanisms that may be involved in the regulation of olfactory ciliary length and highlight potential alternative therapeutic targets for the treatment of ciliopathy-induced dysfunction in the olfactory system and even in other ciliated organ systems.

Molecular Biology Methods in Streptomyces rimosus, a Producer of Oxytetracycline.

Streptomyces rimosus is used for production of the broad-spectrum antibiotic oxytetracycline (OTC). S. rimosus belongs to Actinomyces species, a large group of microorganisms that produce diverse set of natural metabolites of high importance in many aspects of our life. In this chapter, we describe specific molecular biology methods and a classical homologous recombination approach for targeted in-frame deletion of a target gene or entire operon in S. rimosus genome. The presented protocols will guide you through the design of experiment and construction of homology arms and their cloning into appropriate vectors, which are suitable for gene-engineering work with S. rimosus. Furthermore, two different protocols for S. rimosus transformation are described including detailed procedure for targeted gene replacement via double crossover recombination event. Gene deletion is confirmed by colony PCR, and colonies are further characterized by cultivation and metabolite analysis. As the final step, we present in trans complementation of the deleted gene, to confirm functionality of the engineering approach achieved by gene disruption. A number of methodological steps and protocols are optimized for S. rimosus strains including the use of the selected reporter genes. Protocols described in this chapter can be applied for studying function of any individual gene product in diverse OTC-producing Streptomyces rimosus strains.