ABSTRACT
The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation.
ABSTRACT
Vanishing white matter (VWM) is a fatal leukodystrophy caused by recessive mutations in subunits of the eukaryotic translation initiation factor 2B. Currently, there are no effective therapies for VWM. Here, we assessed the potential of adenine base editing to correct human pathogenic VWM variants in mouse models. Using adeno-associated viral vectors, we delivered intein-split adenine base editors into the cerebral ventricles of newborn VWM mice, resulting in 45.9% ± 5.9% correction of the Eif2b5R191H variant in the cortex. Treatment slightly increased mature astrocyte populations and partially recovered the integrated stress response (ISR) in female VWM animals. This led to notable improvements in bodyweight and grip strength in females; however, locomotor disabilities were not rescued. Further molecular analyses suggest that more precise editing (i.e., lower rates of bystander editing) as well as more efficient delivery of the base editors to deep brain regions and oligodendrocytes would have been required for a broader phenotypic rescue. Our study emphasizes the potential, but also identifies limitations, of current in vivo base-editing approaches for the treatment of VWM or other leukodystrophies.
Subject(s)
Dependovirus , Disease Models, Animal , Eukaryotic Initiation Factor-2B , Gene Editing , Leukoencephalopathies , Phenotype , Animals , Mice , Eukaryotic Initiation Factor-2B/genetics , Eukaryotic Initiation Factor-2B/metabolism , Leukoencephalopathies/genetics , Leukoencephalopathies/therapy , Leukoencephalopathies/pathology , Dependovirus/genetics , Humans , Genetic Vectors/genetics , Genetic Vectors/administration & dosage , Female , Mutation , Genetic Therapy/methods , White Matter/pathology , White Matter/metabolism , Astrocytes/metabolismABSTRACT
In this study, the authors compared the efficiency of automated robotic and manual injection methods for the CRISPR-RfxCas13d (CasRx) system for mRNA knockdown and Cas9-mediated DNA targeting in zebrafish embryos. They targeted the no tail (TBXTA) gene as a proof-of-principle, evaluating the induced embryonic phenotypes. Both Cas9 and CasRx systems caused loss of function phenotypes for TBXTA. Cas9 protein exhibited a higher percentage of severe phenotypes compared with mRNA, while CasRx protein and mRNA showed similar efficiency. Both robotic and manual injections demonstrated comparable phenotype percentages and mortality rates. The findings highlight the potential of RNA-targeting CRISPR effectors for precise gene knockdown and endorse automated microinjection at a speed of 1.0 s per embryo as a high-throughput alternative to manual methods.
Subject(s)
CRISPR-Cas Systems , Microinjections , Robotics , Zebrafish , Animals , Zebrafish/genetics , Zebrafish/embryology , CRISPR-Cas Systems/genetics , Microinjections/methods , Robotics/methods , RNA Interference , Embryo, Nonmammalian , Gene Knockdown Techniques/methods , Zebrafish Proteins/genetics , RNA, Messenger/geneticsABSTRACT
Alzheimer's disease is the main cause of aging-associated dementia, for which there is no effective treatment. In this work, we reanalyze the information of a previous genome wide association study, using a new pipeline design to identify novel potential drugs. With this approach, ribonucleoside-diphosphate reductase gene (RRM2B) emerged as a candidate target and its inhibitor, 2', 2'-difluoro 2'deoxycytidine (gemcitabine), as a potential pharmaceutical drug against Alzheimer's disease. We functionally verified the effect of inhibiting the RRM2B homolog, rnr-2, in an Alzheimer's model of Caenorhabditis elegans, which accumulates human Aß1-42 peptide to an irreversible paralysis. RNA interference against rnr-2 and also treatment with 200â ng/ml of gemcitabine, showed an improvement of the phenotype. Gemcitabine treatment increased the intracellular ATP level 3.03 times, which may point to its mechanism of action. Gemcitabine has been extensively used in humans for cancer treatment but at higher concentrations. The 200â ng/ml concentration did not exert a significant effect over cell cycle, or affected cell viability when assayed in the microglia N13 cell line. Thus, the inhibitory drug of the RRM2B activity could be of potential use to treat Alzheimer's disease and particularly gemcitabine might be considered as a promising candidate to be repurposed for its treatment.
Subject(s)
Alzheimer Disease , Caenorhabditis elegans , Deoxycytidine , Disease Models, Animal , Caenorhabditis elegans/drug effects , Alzheimer Disease/drug therapy , Animals , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Deoxycytidine/therapeutic use , Amyloid beta-Peptides/metabolism , Humans , Gemcitabine , Ribonucleoside Diphosphate Reductase/genetics , Ribonucleotide Reductases/antagonists & inhibitors , Ribonucleotide Reductases/metabolism , Adenosine Triphosphate/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , RNA InterferenceABSTRACT
The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. Here we study the nuclease activity of SpG and SpRY by targeting 40 sites in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm could help to predict SpG and SpRY targets with high activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.
Subject(s)
CRISPR-Associated Protein 9 , Gene Editing , Animals , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems/genetics , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Gene Editing/methods , RNA, Guide, Kinetoplastida/genetics , RNA, Messenger , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
CRISPR-Cas systems have been used to induce DNA mutagenesis for gene function discovery. However, the development of tools to eliminate RNAs provides complementary and unique approaches to disrupt gene expression. Here, we present a workflow to perform specific, efficient, and cost-effective mRNA knockdown in zebrafish embryos using our in vivo optimized CRISPR-RfxCas13d (CasRx) system. Although the described protocol focuses on mRNA knockdown in zebrafish embryos, it can also be applied to other vertebrates. For complete details on the use and execution of this protocol, please refer to Kushawah et al. (2020).
Subject(s)
RNA, Guide, Kinetoplastida , Zebrafish , Animals , CRISPR-Cas Systems/genetics , RNA/genetics , RNA, Guide, Kinetoplastida/genetics , RNA, Messenger/genetics , Zebrafish/geneticsABSTRACT
Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to degrade RNA in yeast, plants, and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-RfxCas13d (CasRx) is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that zygotically expressed and maternally provided transcripts are efficiently targeted, resulting in a 76% average decrease in transcript levels and recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish, and mouse embryos. Altogether, our results demonstrate that CRISPR-RfxCas13d is an efficient knockdown platform to interrogate gene function in animal embryos.
Subject(s)
CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing , Gene Expression Regulation, Developmental/genetics , Animals , Gene Editing/methods , HEK293 Cells , Humans , RNA Interference/physiology , RNA, Messenger/geneticsABSTRACT
The awakening of the genome after fertilization is a cornerstone of animal development. However, the mechanisms that activate the silent genome after fertilization are poorly understood. Here, we show that transcriptional competency is regulated by Brd4- and P300-dependent histone acetylation in zebrafish. Live imaging of transcription revealed that genome activation, beginning at the miR-430 locus, is gradual and stochastic. We show that genome activation does not require slowdown of the cell cycle and is regulated through the translation of maternally inherited mRNAs. Among these, the enhancer regulators P300 and Brd4 can prematurely activate transcription and restore transcriptional competency when maternal mRNA translation is blocked, whereas inhibition of histone acetylation blocks genome activation. We conclude that P300 and Brd4 are sufficient to trigger genome-wide transcriptional competency by regulating histone acetylation on the first zygotic genes in zebrafish. This mechanism is critical for initiating zygotic development and developmental reprogramming.
Subject(s)
Embryo, Nonmammalian/metabolism , Embryonic Development , Gene Expression Regulation, Developmental , Genome , Zebrafish Proteins/metabolism , Zebrafish/embryology , Zygote/metabolism , Animals , E1A-Associated p300 Protein/genetics , E1A-Associated p300 Protein/metabolism , Embryo, Nonmammalian/cytology , Regulatory Sequences, Nucleic Acid , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation , Transcriptome , Zebrafish Proteins/genetics , Zygote/cytologyABSTRACT
The impact of the CRISPR-Cas biotechnological systems has recently broadened the genome editing toolbox available to different model organisms further with the addition of new efficient RNA-guided endonucleases. We have recently optimized CRISPR-Cpf1 (renamed Cas12a) system in zebrafish. We showed that (i) in the absence of Cpf1 protein, crRNAs are unstable and degraded in vivo, and CRISPR-Cpf1 RNP complexes efficiently mutagenize the zebrafish genome; and (ii) temperature modulates Cpf1 activity especially affecting AsCpf1, which experiences a reduced performance below 37⯰C. Here, we describe a step-by-step protocol on how to easily design and generate crRNAs in vitro, purify recombinant Cpf1 proteins, and assemble ribonucleoprotein complexes to carry out efficient mutagenesis in zebrafish in a constitutive and temperature-controlled manner. Finally, we explain how to induce Cpf1-mediated homology-directed repair using single-stranded DNA oligonucleotides. In summary, this protocol includes the steps to efficiently modify the zebrafish genome and other ectothermic organisms using the CRISPR-Cpf1 system.
Subject(s)
Bacterial Proteins/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Endonucleases/genetics , Gene Editing/methods , Zebrafish/genetics , Animals , Clostridiales/genetics , DNA End-Joining Repair/genetics , Genome/genetics , RNA, Guide, Kinetoplastida/genetics , Recombinational DNA Repair/geneticsABSTRACT
Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species.
Subject(s)
Bacterial Proteins/metabolism , CRISPR-Cas Systems , Endonucleases/metabolism , Gene Editing/methods , Recombinational DNA Repair , Animals , Bacterial Proteins/genetics , Endonucleases/genetics , Humans , Models, Genetic , Mutagenesis , Temperature , Xenopus/genetics , Zebrafish/geneticsABSTRACT
This protocol describes how to generate and genotype mutants using an optimized CRISPR-Cas9 genome-editing system in zebrafish (CRISPRscan). Because single guide RNAs (sgRNAs) have variable efficiency when targeting specific loci, our protocol starts by explaining how to use the web tool CRISPRscan to design highly efficient sgRNAs. The CRISPRscan algorithm is based on the results of an integrated analysis of more than 1000 sgRNAs in zebrafish, which uncovered highly predictive factors that influence Cas9 activity. Next, we describe how to easily generate sgRNAs in vitro, which can then be injected in vivo to target specific loci. The use of highly efficient sgRNAs can lead to biallelic mutations in the injected embryos, causing lethality. We explain how targeting Cas9 to the germline increases viability by reducing somatic mutations. Finally, we combine two methods to identify F1 heterozygous fish carrying the desired mutations: (i) Mut-Seq, a method based on high-throughput sequencing to detect F0 founder fish; and (ii) a polymerase chain reaction-based fragment analysis method that identifies F1 heterozygous fish characterized by Mut-Seq. In summary, this protocol includes the steps to generate and characterize mutant zebrafish lines using the CRISPR-Cas9 genome engineering system.
Subject(s)
CRISPR-Cas Systems , Gene Editing/methods , Zebrafish/genetics , Animals , Computational Biology/methods , Endonucleases/metabolism , Genotyping Techniques/methods , RNA, Guide, Kinetoplastida/metabolismABSTRACT
The CRISPR-Cas9 system uncovered in bacteria has emerged as a powerful genome-editing technology in eukaryotic cells. It consists of two components-a single guide RNA (sgRNA) that directs the Cas9 endonuclease to a complementary DNA target site. Efficient targeting of individual genes requires highly active sgRNAs. Recent efforts have made significant progress in understanding the sequence features that increase sgRNA activity. In this introduction, we highlight advancements in the field of CRISPR-Cas9 targeting and discuss our web tool CRISPRscan, which predicts the targeting activity of sgRNAs and improves the efficiency of the CRISPR-Cas9 system for in vivo genome engineering.
Subject(s)
CRISPR-Cas Systems , Gene Editing/methods , Endonucleases/metabolism , RNA, Guide, Kinetoplastida/metabolism , Recombination, GeneticABSTRACT
Cellular transitions require dramatic changes in gene expression that are supported by regulated mRNA decay and new transcription. The maternal-to-zygotic transition is a conserved developmental progression during which thousands of maternal mRNAs are cleared by post-transcriptional mechanisms. Although some maternal mRNAs are targeted for degradation by microRNAs, this pathway does not fully explain mRNA clearance. We investigated how codon identity and translation affect mRNA stability during development and homeostasis. We show that the codon triplet contains translation-dependent regulatory information that influences transcript decay. Codon composition shapes maternal mRNA clearance during the maternal-to-zygotic transition in zebrafish, Xenopus, mouse, and Drosophila, and gene expression during homeostasis across human tissues. Some synonymous codons show consistent stabilizing or destabilizing effects, suggesting that amino acid composition influences mRNA stability. Codon composition affects both polyadenylation status and translation efficiency. Thus, the ribosome interprets two codes within the mRNA: the genetic code which specifies the amino acid sequence and a conserved "codon optimality code" that shapes mRNA stability and translation efficiency across vertebrates.
Subject(s)
Codon , Gene Expression Regulation , Protein Biosynthesis , RNA Stability , RNA, Messenger/genetics , Zygote/growth & development , Animals , Drosophila , Humans , Mice , Ribosomes/metabolism , Xenopus , ZebrafishABSTRACT
CRISPR-Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observed that guanine enrichment and adenine depletion increased sgRNA stability and activity, whereas differential sgRNA loading, nucleosome positioning and Cas9 off-target binding were not major determinants. We also identified sgRNAs truncated by one or two nucleotides and containing 5' mismatches as efficient alternatives to canonical sgRNAs. On the basis of these results, we created a predictive sgRNA-scoring algorithm, CRISPRscan, that effectively captures the sequence features affecting the activity of CRISPR-Cas9 in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos 3' UTR led to the generation of maternal-zygotic mutants, as well as increased viability and decreased somatic mutations. These results identify determinants that influence Cas9 activity and provide a framework for the design of highly efficient sgRNAs for genome targeting in vivo.
Subject(s)
Algorithms , CRISPR-Cas Systems , Gene Targeting/methods , RNA, Guide, Kinetoplastida/genetics , 3' Untranslated Regions , Adenine , Animals , Clustered Regularly Interspaced Short Palindromic Repeats , Embryo, Nonmammalian , Female , Gene Expression Regulation , Genetic Engineering/methods , Genome , Guanine , Mutation , Xenopus/genetics , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
MicroRNAs (miRNAs) have been widely studied in order to elucidate their biological functions. MicroRNA microarrays or miRNA overexpression libraries generated by synthesis and cloning of individual miRNAs have been used to study their different roles. In this work, we have developed a novel methodology to express mature miRNAs and other small RNAs from a double convergent RNA polymerase III promoter. We show that the generated miRNAs function similarly to those processed from primary transcripts or pri-miRNAs. This system allowed us to produce a lentiviral library expressing the whole population of small RNAs present in a metastatic cell line. A functional screening using this library led to the identification of hsa-miR-30b and hsa-miR-30c as negative regulators of cell death induced by loss of attachment (anoikis). Importantly, we demonstrated that the acquisition of anoikis resistance via these miRNAs is achieved through down-regulation of caspase 3 expression. Moreover, overexpression of these miRNAs resulted in a decrease of other types of caspase 3-dependent cell death and enhanced the survival of MCF10A acinar cells in morphogenesis assays, suggesting a putative role as oncomirs. In summary, this novel methodology provides a powerful and effective way for identifying novel small RNAs involved in a particular biological process.
Subject(s)
Anoikis/genetics , Caspase 3/genetics , MicroRNAs/genetics , 3' Untranslated Regions , Base Sequence , Binding Sites , Caspase 3/metabolism , Cell Culture Techniques , Cell Shape , Enzyme Repression , Female , Gene Expression , Gene Library , HCT116 Cells , HEK293 Cells , Humans , Mammary Glands, Human/cytology , MicroRNAs/metabolism , Morphogenesis , RNA InterferenceABSTRACT
Pituitary tumor transforming gene 1 (PTTG1), also known as securin, has been implicated in many biological functions, including inhibition of sister chromatid separation, DNA repair, organ development, and regulation of the expression and secretion of angiogenic and metastatic factors. Although most of these functions of securin seem to depend on the localization of PTTG1 in the nucleus of the cell, a fraction of the protein has been also detected in the cytoplasm. Here we demonstrate that, in different cell types, a portion of cytoplasmic PTTG1 is associated with the cis face of the Golgi apparatus and that this localization depends on PTTG1 phosphorylation status. In this organelle, PTTG1 forms a complex with proteins involved in microtubule nucleation, including GM130, AKAP450, and γ-tubulin. RNA interference-mediated depletion of PTTG1 produces a delay in centrosomal and noncentrosomal microtubule nucleation. Cells lacking PTTG1 show severe defects in both cell polarization and migration in wound-healing assays. To our knowledge, this is the first study reporting the role of PTTG1 in microtubule nucleation and cell polarization, two processes directly involved in cell migration. We believe that these findings will contribute to understanding the mechanisms underlying PTTG1-mediated biological functions.
Subject(s)
Cell Movement , Golgi Apparatus/metabolism , Microtubules/metabolism , Neoplasm Proteins/metabolism , A Kinase Anchor Proteins/metabolism , Antibodies, Monoclonal/immunology , Autoantigens/metabolism , Cell Line , Cell Nucleus/metabolism , Cell Polarity , Centrosome , Cytoskeletal Proteins/metabolism , Humans , Membrane Proteins/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Phosphorylation , RNA Interference , RNA, Small Interfering , Securin , Tubulin/metabolism , Wound HealingABSTRACT
Pituitary tumor-transforming gene-1 (PTTG1) is an oncogene highly expressed in a variety of endocrine, as well as nonendocrine-related cancers. Several tumorigenic mechanisms for PTTG1 have been proposed, one of the best characterized being its capacity to act as a transcriptional activator. To identify novel downstream target genes, we have established cell lines with inducible expression of PTTG1 and a differential display approach to analyze gene expression changes after PTTG1 induction. We identified dlk1 (also known as pref-1) as one of the most abundantly expressed PTTG1 targets. Dlk1 is known to participate in several differentiation processes, including adipogenesis, adrenal gland development, and wound healing. Dlk1 is also highly expressed in neuroendocrine tumors. Here, we show that PTTG1 overexpression inhibits adipogenesis in 3T3-L1 cells and that this effect is accomplished by promoting the stability and accumulation of Dlk1 mRNA, supporting a role for PTTG1 in posttranscriptional regulation. Moreover, both pttg1 and dlk1 genes show concomitant expression in fetal liver and placenta, as well as in pituitary adenomas, breast adenocarcinomas, and neuroblastomas, suggesting that PTTG1 and DLK1 are involved in cell differentiation and transformation.
Subject(s)
Adipocytes/cytology , Cell Differentiation , Cell Transformation, Neoplastic/pathology , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/genetics , Neoplasm Proteins/metabolism , 3T3-L1 Cells , Adipocytes/metabolism , Animals , Calcium-Binding Proteins , Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic , HCT116 Cells , Hepatocyte Nuclear Factor 3-beta/metabolism , Humans , Membrane Proteins/metabolism , Mice , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , Securin , Signal TransductionABSTRACT
In Trichoderma harzianum CECT 2413 external pH regulates essential functions such as growth rate, sporulation, cell and colony morphology, pattern of secreted proteins, gene expression or mycoparasitism-related enzymatic activities. pH regulation is mediated by the transcriptional factor Pac1 (homologous to PacC regulator in other fungi), encoded by pac1 whose expression increases with pH. Two pac1 mutants have been obtained from CECT 2413: P2.32, that possesses an allele of pac1 active at any pH, and R13, that does not express pac1 due to interferent RNA. Cell morphology, sporulation and growth rate are optimal for strain P2.32 at pH 7.5 and for strain R13 at pH 3.0, mimicking alkaline and acidic conditions, respectively. Pac1 regulates some genes involved in antagonism: chit42 chitinase, papA protease, gtt1 glucose permease, and qid74 cell wall protein. Furthermore, Pac1 modulates T. harzianum antifungal activity since wild type and mutants inhibit several phytopathogenic fungal strains at various degrees in different assays.
Subject(s)
Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Trichoderma/genetics , Fungal Proteins/metabolism , Fungal Proteins/physiology , Hydrogen-Ion Concentration , Mutation , RNA, Fungal/genetics , RNA, Fungal/metabolism , Spores, Fungal/genetics , Spores, Fungal/growth & development , Trichoderma/physiologyABSTRACT
Trichoderma is widely used as biocontrol agent against phytopathogenic fungi, and as biofertilizer because of its ability to establish mycorriza-like association with plants. The key factor to the ecological success of this genus is the combination of very active mycoparasitic mechanisms plus effective defense strategies induced in plants. This work, different from most of the studies carried out that address the attacking mechanisms, focuses on elucidating how Trichoderma is able to tolerate hostile conditions. A gene from Trichoderma harzianum CECT 2413, qid74, was strongly expressed during starvation of carbon or nitrogen sources; it encoded a cell wall protein of 74kDa that plays a significant role in mycelium protection. qid74 was originally isolated and characterized, in a previous work, by a differential hybridization approach under simulated mycoparasitism conditions. Heterologous expression of Qid74 in Saccharomyces cerevisiae indicated that the protein, located in the cell wall, interfered with mating and sporulation but not with cell integrity. The qid74 gene was disrupted by homologous recombination and it was overexpressed by isolating transformants selected for the amdS gene that carried several copies of qid74 gene under the control of the pki promoter. Disruptants and transformants showed similar growth rate and viability when they were cultivated in different media, temperatures and osmolarities, or were subjected to different abiotic stress conditions. However, disruptants produced about 70% mass yield under any condition and were substantially more sensitive than the wild type to cell wall degradation by different lytic preparations. Transformants had similar mass yield and were more resistant to lytic enzymes but more sensitive to copper sulfate than the wild type. When experiments of adherence to hydrophobic surfaces were carried out, the disruptants had a reduced capacity to adhere, whereas that capacity in the overproducer transformants was slightly higher than that of the wild type. Results point to a significant role for Qid74 both in cell wall protection and adhesion to hydrophobic surfaces.
Subject(s)
Fungal Proteins/physiology , Membrane Proteins/physiology , Trichoderma/physiology , Cell Wall/metabolism , Cell Wall/physiology , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression , Hydrophobic and Hydrophilic Interactions , Membrane Proteins/genetics , Membrane Proteins/metabolism , Pest Control, Biological/methods , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Trichoderma/genetics , Trichoderma/growth & developmentABSTRACT
Three procedures were used to obtain new Saccharomyces cerevisiae baker's yeasts with increased storage stability at -20, 4, 22, and 30 degrees C. The first used mitochondria from highly ethanol-tolerant wine yeast, which were transferred to baker's strains. Viability of the heteroplasmons was improved shortly after freezing. However, after prolonged storage, viability dramatically decreased and was accompanied by an increase in the frequency of respiratory-deficient (petite) mutant formation. This indicated that mitochondria were not stable and were incompatible with the nucleus. The strains tested regained their original resistance to freezing after recovering their own mitochondria. The second procedure used hybrid formation after protoplast fusion and isolation on selective media of fusants from baker's yeast meiotic products resistant to parafluorphenylalanine and cycloheximide, respectively. No hybrids were obtained when using the parentals, probably due to the high ploidy of the baker's strains. Hybrids obtained from nonisogenic strains manifested in all cases a resistance to freezing intermediate between those of their parental strains. Hybrids from crosses between meiotic products of the same strain were always more sensitive than their parentals. The third method was used to develop baker's yeast mutants resistant to 2-deoxy-d-glucose (DOG) and deregulated for maltose and sucrose metabolism. Mutant DOG21 displayed a slight increase in trehalose content and viability both in frozen doughs and during storage at 4 and 22 degrees C. This mutant also displayed a capacity to ferment, under laboratory conditions, both lean and sweet fresh and frozen doughs. For industrial uses, fermented lean and sweet bakery products, both from fresh and frozen doughs obtained with mutant DOG21, were of better quality with regard to volume, texture, and organoleptic properties than those produced by the wild type.