Reverse Genetics Tool Research Articles

Sequence-specific nucleases as tools for enhancing disease resistance in crops

Genome editing technologies, such as CRISPR/Cas, have recently become valuable tools for plant reverse genetics as well as crop improvement, including enhancement of disease resistance. Targeting susceptibility (S) genes by genome editing has proven to be a viable strategy for generating resistance to both bacterial and fungal pathogens in various crops. Examples include generating loss-of-function mutations in promoter elements of the SWEET S genes, which are targeted by transcription activator-like effectors secreted by many phytopathogenic Xanthomonas bacteria, as well as in the conserved MLO locus that confers susceptibility to powdery mildew fungal pathogens in many monocots and dicots. In addition to genome editing applications, CRISPR/Cas systems can be used as means of defending plants against viruses via targeting viral genomic DNA or RNA. Genome editing is therefore a highly promising approach that enables engineering disease resistance to various plant pathogens directly in elite cultivar background in a highly precise manner. Unlike conventional crop breeding, genome editing approaches are not relying on lengthy and laborious crosses/back-crosses involving parental and progeny lines and can significantly shorten the breeding timeline. Taking into account the high potential of genome editing technologies for both basic and applied plant science, the recent decision of the European Court of Justice to define transgene-free genetically edited crops as GMOs is, clearly, a backward step for the EU.

Development of Targeting Induced Local Lesions IN Genomes (TILLING) Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis.

Targeting Induced Local Lesions IN Genomes (TILLING) is a powerful reverse genetics tool that includes chemical mutagenesis and detection of sequence variation in target genes. TILLING is a highly valuable functional genomics tool for gene validation, especially in small grains in which transformation-based approaches hold serious limitations. Developing a robust mutagenized population is key to determining the efficiency of a TILLING-based gene validation study. A TILLING population with a low overall mutation frequency indicates that an impractically large population must be screened to find desired mutations, whereas a high mutagen concentration leads to high mortality in the population, leading to an insufficient number of mutagenized individuals. Once an effective population is developed, there are multiple ways to detect mutations in a gene of interest, and the choice of platform depends upon the experimental scale and availability of resources. The Cel-1 assay and agarose gel-based approach for mutant identification is convenient, reproducible, and a less resource-intensive platform. It is advantageous in that it is simple, requiring no computational knowledge, and it is especially suitable for validation of a small number of genes with basic lab equipment. In the present article, described are the methods for development of a good TILLING population, including preparation of the dosage curve, mutagenesis and maintenance of the mutant population, and screening of the mutant population using the PCR-based Cel-1 assay.

Genome-wide identification and analysis of genes associated with RNA interference in Bemisia tabaci.

As a method of RNA-mediated gene silencing, RNA interference (RNAi) is a useful reverse genetic tool with which to study gene function, and holds great promise for pest management. Bemisia tabaci is a cosmopolitan pest that causes extensive damage to crops. The mechanism underlying RNAi efficiency in B. tabaci is not well known. We identified and analyzed candidate genes in the RNAi pathway to understand the RNAi mechanism and provide a basis for the application of RNAi in pest management. We identified 33 genes putatively involved in the RNAi pathway from the B. tabaci Q genome. Phylogenetic and structural analyses confirmed the characteristics of these genes. Furthermore, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and transcriptomic analysis profiled gene expression patterns during different developmental stages. Gene expression levels estimated by qRT-PCR and RNA-seq analyses were significantly correlated. Moreover, gene functions were verified by RNAi. When accompanied by knockdown of AGO2, Dicer2 and Sid1, the efficiency of CYP6DB3 RNAi decreased correspondingly. In this study, we annotated and validated genes involved in B. tabaci RNAi. A better understanding of the building blocks of the RNAi process in B. tabaci facilitates integration of this novel biotechnology into the management of this emerging pest, either directly or indirectly. © 2019 Society of Chemical Industry.

An efficient TILLING platform for cultivated tobacco.

Targeting-induced local lesions in genomes (TILLING) is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants. Although TILLING has been developed for many diploid plants, the technology has been used in very few polyploid species due to their genomic complexity. Here, we established an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco (Nicotiana tabacum L.) using an ethyl methanesulfonate (EMS)-mutagenized population of 1,536 individuals. We optimized the procedures for endonuclease preparation, leaf tissue sampling, DNA extraction, normalization, pooling, PCR amplification, heteroduplex formation, and capillary electrophoresis. In a test screen using seven target genes with eight PCR fragments, we obtained 118 mutants. The mutation density was estimated to be approximately one mutation per 106 kb on average. Phenotypic analyses showed that mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc, which was confirmed independently using CRISPR/Cas9 to generate knockout mutants. Our results demonstrate that this powerful TILLING platform (available at http://www.croptilling.org) can be used in tobacco to facilitate functional genomics applications.

RNA Interference and Small RNA Analysis.

Small silencing RNAs have provided powerful reverse genetics tools and have opened new areas of research. This introduction describes the use of RNAi to suppress expression of individual genes for loss-of-function analysis. It also summarizes methods for measuring specific and global changes in small RNA expression, as well as methods to inhibit the function of individual endogenous small RNA species such as miRNAs.

RNAi-mediated knockdown of daf-12 in the model parasitic nematode Strongyloides ratti.

The gene daf-12 has long shown to be involved in the dauer pathway in Caenorhabditis elegans (C. elegans). Due to the similarities of the dauer larvae of C. elegans and infective larvae of certain parasitic nematodes such as Strongyloides spp., this gene has also been suspected to be involved in the development of infective larvae. Previous research has shown that the application of dafachronic acid, the steroid hormone ligand of DAF-12 in C. elegans, affects the development of infective larvae and metabolism in Strongyloides. However, a lack of tools for either forward or reverse genetics within Strongyloides has limited studies of gene function within these important parasites. After determining whether Strongyloides had the requisite proteins for RNAi, we developed and report here the first successful RNAi by soaking protocol for Strongyloides ratti (S. ratti) and use this protocol to study the functions of daf-12 within S. ratti. Suppression of daf-12 in S. ratti severely impairs the formation of infective larvae of the direct cycle and redirects development towards the non-infective (non-dauer) free-living life cycle. Further, daf-12(RNAi) S. ratti produce slightly but significantly fewer offspring and these offspring are developmentally delayed or incapable of completing their development to infective larvae (L3i). Whilst the successful daf-12(RNAi) L3i are still able to infect a new host, the resulting infection is less productive and shorter lived. Further, daf-12 knockdown affects metabolism in S. ratti resulting in a shift from aerobic towards anaerobic fat metabolism. Finally, daf-12(RNAi) S. ratti have reduced tolerance of temperature stress.

RNA interference-mediated knockdown of voltage-gated sodium channel (MpNav) gene causes mortality in peach-potato aphid, Myzus persicae

Voltage-gated sodium channels (VGSC) are transmembrane proteins that generate an action potential in excitable cells and play an essential role in neuronal signaling. Since VGSCs play a crucial role in nerve transmission they have become primary targets for a broad range of commercial insecticides. RNA interference (RNAi) is a valuable reverse genetics tool used in functional genomics, but recently, it has also shown promise as a novel agent that could be used to control agricultural insect pests. In this study, we targeted the VGSC (MpNav) gene in the peach-potato aphid Myzus persicae, by oral feeding of artificial diets mixed with dsRNAs. Knock-down of MpNav gene expression caused up to 65% mortality in 3rd instar nymphs. Moreover, significantly lower fecundity and longevity was observed in adult aphids that had been fed with dsMpNav solution at the nymphal stage. Analysis of gene expression by qRT-PCR indicated that the aphid mortality rates and the lowered fecundity and longevity were attributable to the down-regulation of MpNav by RNAi. Taken together, our results show that MpNav is a viable candidate target gene for the development of an RNAi-based bio-aphicide.

The ecdysone receptor complex is essential for the reproductive success in the female desert locust, Schistocerca gregaria

Ecdysteroid hormones influence the development and reproduction of arthropods by binding a heterodimeric complex of nuclear receptors, the ecdysone receptor (EcR) and the retinoid-X-receptor/ultraspiracle (RXR/USP). Here, we report on the in vivo role(s) of the ecdysone receptor complex, SchgrEcR/SchgrRXR, in the female reproductive physiology of a major phytophagous pest insect, i.e. the desert locust, Schistocerca gregaria. Tissue and temporal distribution profiles were analysed during the first gonadotrophic cycle of adult female locusts. RNA interference was used as a reverse genetics tool to investigate the in vivo role of the ecdysone receptor complex in ovarian maturation, oogenesis, fertility and fecundity. We discovered that silencing the ecdysone receptor complex in S. gregaria resulted in impaired ovulation and oviposition, indicative for a crucial role of this complex in chorion formation. We also found evidence for a feedback of SchgrEcR/SchgrRXR on juvenile hormone biosynthesis by the corpora allata. Furthermore, we observed a tissue-dependent effect of the SchgrEcR/SchgrRXR knockdown on the transcript levels of the insulin receptor and neuroparsin 3 and 4. The insulin receptor transcript levels were upregulated in the brain, but not the fat body and gonads. Neuroparsins 3 and 4 transcript levels were down regulated in the brain and fat body, but not in the gonads.

The Cricket Gryllus bimaculatus: Techniques for Quantitative and Functional Genetic Analyses of Cricket Biology.

All extant species are an outcome of nature's "experiments" during evolution, and hence multiple species need to be studied and compared to gain a thorough understanding of evolutionary processes. The field of evolutionary developmental biology (evo-devo) aspires to expand the number of species studied, because most functional genetic studies in animalshave been limited to a small number of "traditional" model organisms, many of which belong to the same phylum (Chordata). The phylum Arthropoda, and particularly its component class Insecta, possesses many important characteristics that are considered favorable and attractive for evo-devo research, including an astonishing diversity of extant species and a wide disparity in body plans. The development of the most thoroughly investigated insect genetic model system to date, the fruit fly Drosophila melanogaster (a holometabolous insect), appears highly derived with respect to other insects and indeed with respect to most arthropods. In comparison, crickets (a basally branching hemimetabolous insect lineage compared to the Holometabola) are thought to embody many developmental features that make them more representative of insects. Here we focus on crickets as emerging models to study problems in a wide range of biological areas and summarize the currently available molecular, genomic, forward and reverse genetic, imaging and computational tool kit that has been established or adapted for cricket research. With an emphasis on the cricket species Gryllus bimaculatus, we highlight recent efforts made by the scientific community in establishing this species as a laboratory model for cellular biology and developmental genetics. This broad toolkit has the potential to accelerate many traditional areas of cricket research, including studies of adaptation, evolution, neuroethology, physiology, endocrinology, regeneration, and reproductive behavior. It may also help to establish newer areas, for example, the use of crickets as animal infection model systems and human food sources.

Applications of Virus Induced Gene Silencing (VIGS) in Plant Functional Genomics Studies

Virus induced gene silencing (VIGS) is an effective technology that exploits an antiviral defense mechanism in plants. It is a recently developed gene transcript suppression technique for characterizing the function of plant genes. VIGS is rapid, efficient and specific system for transient gene silencing. The major steps in VIGS includes; engineering viral genomes to the appropriate viral vector to incorporate fragments of host genes that are targeted to be silenced, infecting the appropriate plant hosts and silencing the target genes as part of the defense mechanism of the plant against virus infection. The VIGS vector is a recombinant virus engineered to be able to carry a piece an endogenous gene from the host. During infection with the modified vector, the host’s defense reaction will be induced against the cloned host gene; a loss of function phenotype makes it possible to identify the function of the gene. The recombinant virus is introduced into plant cells through Agrobacterium tumefaciens mediated transient expression or in vitro transcribed RNA inoculation or direct DNA inoculation. The Trans gene is amplified along with the viral RNA by RNA dependent RNA polymerase generating dsRNA molecules. dsRNA is the triggering molecule of Post transcriptional gene silencing. VIGS as a reverse genetics tool for functional genomics studies presenting several advantages. Despite its great potential, many limitations remain to be overcome. In this review, the molecular mechanism in VIGS technology, its advanced application in plant functional genomics studies and the major limitation and potential future prospects were briefly discussed.

RNA interference of muscle actin and ATP synthase beta increases mortality of the phytoplasma vector Euscelidius variegatus.

RNA interference (RNAi) techniques have emerged as powerful tools to develop novel management strategies for the control of insect pests. The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma and a laboratory vector of Flavescence dorée phytoplasma. Phytoplasmas are insect-borne bacterial plant pathogens that cause economically relevant crop losses worldwide. In this study, we demonstrated that microinjection of muscle actin and ATP synthase β double-stranded (ds)RNAs into adult insects caused an exponential reduction in the expression of both genes, which began within 72 h of dsRNA administration and lasted for 14 days, leading to almost complete silencing of the target genes. Such silencing effects on muscle actin expression appeared to be both time- and dose-dependent. Our results also showed that the knockdown of both genes caused a significant decrease in survival rates in comparison with green fluorescent protein (GFP) dsRNA-injected control insects. The effectiveness of RNAi-based gene silencing in E. variegatus guarantees the availability of a powerful reverse genetic tool for the functional annotation of its genes and the identification of those potentially involved in the interaction with phytoplasmas. In addition, this study demonstrated that muscle actin and ATP synthase β may represent candidate genes for RNAi-based control of E. variegatus. © 2018 Society of Chemical Industry.

Tobacco Rattle Virus-Induced Gene Silencing in Cotton.

Virus-induced gene silencing (VIGS), as a tool for plant reverse genetics, has been widely used in cotton for target gene function analysis. Compared with genetically transformed plants, the target gene expression level is reduced in the newly emerged leaves and can carry out phenotype identification after a few weeks of Agrobacterium infiltration. In this chapter, we describe a detailed protocol for Agrobacterium-mediated TRV-VIGS system for cotton gene function studies, with focus on designing primers, constructing TRV-target gene vectors via homologous recombination method, preparing and infiltrating Agrobacterium with TRV-VIGS, and identifying target gene silencing.

Gene Silencing: The Mechanism to Down Regulate the Target Gene

Gene silencing is one of the most efficient and promising functional genomics tools which down regulates the expression of a gene in a very precise manner and has significant impact on crop improvement. Silencing of a target gene can be achieved at two levels; transcriptional and post-transcriptional stages. In broader sense, transcriptional gene silencing occurs through the repression of the process of transcription while the post transcriptional gene silencing can occur through the degradation of the mRNA. Small RNA molecules like siRNAs & miRNAs are key players which mediate the mechanism of gene silencing. These small RNA molecules are generated through Dicer digestion of the endogenous or exogenous dsRNA by which RISC (RNA induced gene silencing complex) is activated which inhibits expression of a gene. Apart from these small RNA molecules, viruses can also accomplish gene silencing which is known as VIGS (Virus induced gene silencing). VIGS consists of cloning and inserting plant endogenous gene sequences in recombinant viral vectors, which are then inoculated in plants, triggering RNA interference. Gene silencing as reverse genetic tool, has been exploited successfully to build up the resistant against biotic (insect/pest, nematodes, viruses etc.) and abiotic stresses (drought, salinity, frost etc.), and for the enhancement of yield and quality parameters. With the development of cutting edge biotechnological tools and techniques, it is now affordable to knock down/down regulate the expression of any undesirable gene. Ultimately this kind of technology can be important to global food security and sustainability.

Forward and reverse genetics approaches for combined stress tolerance in rice

Climate change impact on global agricultural food production has been evident in the past few decades. Abiotic factors such as heat, drought, and salinity share a major proportion of crop yield losses and posing a serious threat to global food security. Developing climate resilient crops has become a frontier area of basic plant science and agricultural research. Persistent efforts by scientists to understand crop responses under natural environment and progress in the field of genomics and phenomics has provided unprecedented pace to crop development programs. Rice is the most important cereal crop and staple food for more than 3 billion people worldwide. Heat, drought and salinity stress are the major constraints for global rice production. Hence, efforts are warranted to develop climate-resilient rice cultivars that can produce substantially under different abiotic stresses. Crop plants seldom face single stress in the natural environment. Indeed, heat and drought or drought and salinity are documented as very obvious combinations suggesting multiple stress tolerance as an important breeding target. Forward and reverse genetic tools could effectively contribute towards achieving the target food production to feed the future population despite limiting resources and unfavorable climatic conditions. Genetic approaches adopted for crop improvement programs categorized as forward and reverse genetics are discussed highlighting their potential benefits for tailoring stress tolerant cultivars.

Creating Targeted Gene Knockouts in Barley Using CRISPR/Cas9.

Knockout mutants are an invaluable reverse genetics tool which have not been well developed in crop species compared to models like Arabidopsis. However, the emergence of CRISPR/Cas9 has changed this situation making the generation of such mutants accessible to many crops including barley. A single T-DNA construct can be transformed into barley immature embryos and stable transgenic lines regenerated through tissue culture which contain targeted mutations. Mutations are detected in T0 plants and go on in subsequent T1 and T2 generations to segregate from T-DNA, leaving lines which are non-transgenic and carrying a variety of mutations at the target locus. These mutations can be targeted to a particular gene of interest in order to bring about a loss of function creating a knockout mutant.

RNAi Screening: Automated High-Throughput Liquid RNAi Screening in Caenorhabditis elegans.

RNAi is a powerful reverse genetics tool that has revolutionized genetic studies in model organisms. The bacteriovorous nematode Caenorhabditis elegans can be genetically manipulated by feeding it an Escherichia coli strain that expresses a double-stranded RNA (dsRNA) corresponding to a C. elegans gene, which leads to systemic silencing of the gene. This unit describes protocols for performing an automated high-throughput RNAi screen utilizing a full-genome C. elegans RNAi library. The protocols employ liquid-handling robotics and 96-well plates. © 2018 by John Wiley & Sons, Inc.

Genomics, GPCRs and new targets for the control of insect pests and vectors.

The pressing need for new pest control products with novel modes of action has spawned interest in small molecules and peptides targeting arthropod GPCRs. Genome sequence data and tools for reverse genetics have enabled the prediction and characterization of GPCRs from many invertebrates. We review recent work to identify, characterize and de-orphanize arthropod GPCRs, with a focus on studies that reveal exciting new functional roles for these receptors, including the regulation of metabolic resistance. We explore the potential for insecticides targeting Class A biogenic amine-binding and peptide-binding receptors, and consider the innovation required to generate pest-selective leads for development, within the context of new PCR-targeting products to control arthropod vectors of disease.

Molecular mechanisms influencing efficiency of RNA interference in insects.

RNA interference (RNAi) is an endogenous, sequence-specific gene-silencing mechanism elicited by small RNA molecules. RNAi is a powerful reverse genetic tool, and is currently being utilized for managing insects and viruses. Widespread implementation of RNAi-based pest management strategies is currently hindered by inefficient and highly variable results when different insect species, strains, developmental stages, tissues, and genes are targeted. Mechanistic studies have shown that double-stranded ribonucleases (dsRNases), endosomal entrapment, deficient function of the core machinery, and inadequate immune stimulation contribute to limited RNAi efficiency. However, a comprehensive understanding of the molecular mechanisms limiting RNAi efficiency remains elusive. Recent advances in dsRNA stability in physiological tissues, dsRNA internalization into cells, the composition and function of the core RNAi machinery, as well as small-interfering RNA/double-stranded RNA amplification and spreading mechanisms are reviewed to establish a global understanding of the obstacles impeding wider understanding of RNAi mechanisms in insects. © 2018 Society of Chemical Industry.

Derivation and characterization of a California grunion (Leuresthes tenuis) embryonic cell line

An immortalized cell line was established from newly hatched embryos of the California grunion (Leuresthes tenuis), a silverside fish, to generate a tool for mechanistic studies explaining the effect of environmental stressors on proteome dynamics. In this oviparous species, fertilization occurs terrestrially, and embryos develop in beach sand until environmentally-cued hatching. Grunion eggs and embryos are subject to multiple environmental stressors including changes in salinity, desiccation, heat, and pollutant runoff, which can reduce fertilization, embryonic development, and hatching success [1–3]. Cell lines derived from grunion embryo primary tissue explants thus provide a valuable tool for high-throughput reverse genetics (e.g., high-throughput enhancer trap assays, knock-out studies, and targeted mutagenesis) and proteomics approaches to enable mechanistic explanations of cellular and molecular responses to environmental stressors. Two parallel cell lines were derived from the same whole grunion embryonic tissue and have been maintained now over 10 passages at a 1:6 splitting ratio. Both cell lines display an epithelial-like phenotype (cuboidal, monolayer, optically dense). A time course experiment was conducted to document effects of cell immortalization on the proteome of both cell lines sampled at each passage. Data-dependent mass spectrometry acquisition of peptide spectra and de novo sequencing were used to annotate spectral libraries with protein identification information. Spectral libraries were then used to construct and validate target sets that enable quantitation of proteome dynamics with data-independent acquisition (cDIA) and Skyline. Passage-dependent changes in proteome dynamics were identified by cDIA target set quantitation with Skyline and correspond to morphological changes in cell types over time. Support or Funding Information Supported by NSF Grant IOS-1355098. Fibroblast and epithelial-like cells derived from grunion after the first passage (A) and third passage (B). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development and Phenotypic Screening of an Ethyl Methane Sulfonate Mutant Population in Soybean.

Soybean is an important oil-producing crop in the Fabaceae family and there are increasing demands for soybean oil and other soybean products. Genetic improvement of soybean is needed to increase its production. In order to provide genetic diversity and resources for identifying important genes, a new ethyl methane sulfonate (EMS) mutagenized soybean population was generated using the newly released germplasm, JTN-5203 (maturity group V). Treatment of soybean seeds with 60 mM EMS concentration was found to be suitable for inducing mutation. A total of 1,820 M1 individuals were produced from 15,000 treated seeds. The resulting M2 population was planted in the field for phenotyping. After harvest, seed traits including total oil, protein, starch, moisture content, fatty acid and amino acid compositions were measured by NIR. Phenotypic variations observed in this population include changes in leaf morphology, plant architecture, seed compositions, and yield. Of most interest, we identified plants with increased amounts of total protein (50% vs. 41% for control) and plants with higher amounts of total oil (25% vs. 21.2% control). Similarly, we identified plants with increases in oleic acid content and decreases in linoleic acid and linolenic acid. This EMS mutant population will be used for further studies including screening for various traits such as amino acid pathways, allergens, phytic acids, and other important soybean agronomic traits. In addition, these mutant individuals will be evaluated in the next generation to assess the heritability. Beneficial traits from these mutants can be exploited for future soybean breeding programs. This germplasm can also be used for discovering novel mutant alleles and for functional gene expression analysis using reverse genetics tools such as TILLING.