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Biological control is the control of disease by the application of biological agents to a host animal or plant that prevents the development of disease by a pathogen. With regard to plant diseases the biocontrol agents are usually bacterial or fungal strains isolated from the endosphere or rhizosphere. Viruses can also be used as biocontrol agents and there is a resurgent interest in the use of bacterial viruses for control of plant diseases. The degree of disease suppression achieved with biological agents can be comparable to that achieved with chemicals. Our understanding of the ways in which biocontrol agents protect plants from disease has developed considerably in recent years with the application of genomics and genetic modification techniques. We have uncovered mechanisms by which biocontrol agents interact with the host plant and other members of the microbial community associated with the plant. Understanding these mechanisms is crucial to the isolation of effective biocontrol agents and the development of biocontrol strategies for plant diseases. This review looks at recent developments in our understanding of biocontrol agents for plant diseases and how they work.

Ecological interactions between different species of yeasts have been observed and described extensively, but the mechanisms of interaction remain poorly understood. A hindrance to the characterization of multispecies yeast ecosystems is the lack of accurate methods for rapid real-time analysis of population dynamics in synthetic multispecies consortia. Here, we sought to accelerate and improve the sensitivity of ecological modelling and characterization of a synthetic yeast ecosystem by developing a flow cytometry–based method that tracks and sorts fluorescently tagged individual yeast species in real time during growth in model multispecies consortia. A protocol for integrative genetic modification of non-conventional yeasts was developed. The application of the method was demonstrated in a model four-species synthetic wine-yeast ecosystem that consisted of species commonly isolated from natural wine fermentations. The data show that this method allows for rapid generation of meaningful ecological data that contributes to our understanding of multispecies synthetic yeast ecosystems. Furthermore, interspecies interactions have been shown to impact the evolution of yeasts in natural ecosystems, and this platform will provide an ideal tool to better evaluate the impact of biotic selection pressures.Key Points• Fluorescent labelling of yeast species in a consortium for multicolour flow cytometry• Method developed to track population dynamics of multispecies yeast consortia• Enables real-time visualization, manipulation and response analyses of population dynamics• Produces accurate, reproducible data with powerful visual analyses potential at a rapid rate

This review recapitulates the history, important milestones, the current status, and the perspectives of the pea (Pisum sativum L.) transformation as a tool for pea crop breeding. It summarises the developments of the pea transformation from the first methodological experiments to achieving the complete transformation and regeneration of genetically modified (GM) plants, transformation with the first genes of interest (GOI), to recent techniques of targeted genome editing. We show how recent biotechnological methods and genetic engineering may contribute to pea breeding in order to speed up the breeding process and for the creation of new pea cultivars. The focus is laid on genetic engineering which represents an excellent technology to enhance the pea gene pool with genes of interest which are not naturally present in the pea genome. Different methods of pea transformation are mentioned, as well as various GOI that have been used for pea transformation to date, all aimed at improving transgenic pea traits. Tolerance to herbicides or resistance to viruses, fungal pathogens, and insect pests belong, among others, to the pea traits that have already been modulated by methods of genetic engineering. The production of phytopharmaceuticals is also an important chapter in the use of genetically modified peas. We compare different methods of introducing transgenes to peas and also the usage of different selective and reporter genes. The transformation of other major legumes (soybeans, beans) is marginally mentioned. The effect of genetically modified (GM) peas on animal health (feeding tests, allergenicity) is summarised, the potential risks and benefits of pea modification are evaluated and also the prime expectations of GM peas and the real current state of this technology are compared. Unfortunately, this technology or, more precisely, the products created by this technology are under strict (albeit not scientifically-based) legislative control in the European Union.

Neoantigens, which are derived from tumour-specific protein-coding mutations, are exempt from central tolerance, can generate robust immune responses 1 , 2 and can function as bona fide antigens that facilitate tumour rejection 3 . Here we demonstrate that a strategy that uses multi-epitope, personalized neoantigen vaccination, which has previously been tested in patients with high-risk melanoma 4 – 6 , is feasible for tumours such as glioblastoma, which typically have a relatively low mutation load 1 , 7 and an immunologically ‘cold’ tumour microenvironment 8 . We used personalized neoantigen-targeting vaccines to immunize patients newly diagnosed with glioblastoma following surgical resection and conventional radiotherapy in a phase I/Ib study. Patients who did not receive dexamethasone—a highly potent corticosteroid that is frequently prescribed to treat cerebral oedema in patients with glioblastoma—generated circulating polyfunctional neoantigen-specific CD4 + and CD8 + T cell responses that were enriched in a memory phenotype and showed an increase in the number of tumour-infiltrating T cells. Using single-cell T cell receptor analysis, we provide evidence that neoantigen-specific T cells from the peripheral blood can migrate into an intracranial glioblastoma tumour. Neoantigen-targeting vaccines thus have the potential to favourably alter the immune milieu of glioblastoma. Neoantigen-targeting vaccines are a feasible therapy for tumours with a low mutation burden and immunologically ‘cold’ tumour microenvironment, as neoantigen-specific T cells from the peripheral blood migrate into intracranial glioblastoma, thereby altering the immune milieu of the glioblastoma.

This study aimed to prepare the sugar industry for the possible introduction of genetically modified (GM) sugarcane and derived retail sugar products and to address several potential public concerns regarding the characteristics and safety of these products. GM sugarcane lines with integrated Cry1Ab and EPSPS foreign genes were used for GM sugar production. Traditional PCR, real-time fluorescent quantitative PCR (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were performed in analyzing leaves, stems, and other derived materials during sugar production, such as fibers, clarified juices, filter mud, syrups, molasses, and final GM sugar product. The toxicity of GM sugar was examined with a feeding bioassay using Helicoverpa armigera larvae. PCR and RT-qPCR results showed that the leaves, stems, fibers, juices, syrups, filter mud, molasses, and white granulated sugar from GM sugarcane can be distinguished from those derived from non-GM sugarcane. The RT-qPCR detection method using short amplified product primers was more accurate than the traditional PCR method. Molecular analysis results indicated that trace amounts of DNA residues remain in GM sugar, and thus it can be accurately characterized using molecular analysis methods. ELISA results showed that only the leaves, stems, fibers, and juices sampled from the GM sugarcane differed from those derived from the non-GM sugarcane, indicating that filter mud, syrup, molasses, and white sugar did not contain detectable Cry1Ab and EPSPS proteins. Toxicity analysis showed that the GM sugar was not toxic to the H. armigera larvae. The final results showed that the GM sugar had no active proteins despite containing trace amounts of DNA residues. This finding will help to pave the way for the commercialization of GM sugarcane and production of GM sugar.

Microbial cell surface display has attracted greater attention than ever and has numerous potential applications in biotechnology. With the safety and probiotic properties, lactic acid bacteria (LAB) have been used widely in food and industrial applications. In order to circumvent using genetically modified microorganisms which face low public acceptance and severe regulatory scrutiny, surface-engineered LAB without genetical modification are more preferred. According to the way used to obtain the fusion protein containing the passenger molecule and anchoring domain, the genetic or chemical approaches can be used to construct these surface-engineered LAB. In addition to the viable wide-type LAB, non-living bacterial-like particles (BLP) can be attached by these fusion proteins added from outside. Compared to the living LAB, BLP have a higher binding capacity and less anticarrier response. Mucosal vaccines are the predominant application of these surface-engineered LAB with no genetical modification.

Actinobacteria are extremely important to human health, agriculture, and forests. Because of the vast differences of the characteristics of Actinobacteria, a lot of genetic tools have been developed for efficiently manipulating the genetics. Although there are a lot of successful examples of engineering Actinobacteria, they are still more difficult to be genetically manipulated than other model microorganisms such as Saccharomyces cerevisiae , Escherichia coli , and Bacillus subtilis etc. due to the diverse genomics and biochemical machinery. Here, we review the methods to introduce heterologous DNA into Actinobacteria and the available genetic modification tools. The trends and problems existing in engineering Actinobacteria are also covered.

Flooding negatively affects the growth of soybeans. Recently, omic approaches have been used to study abiotic stress responses in plants. To explore flood-tolerant genes in soybeans, an integrated approach of proteomics and computational genetic modification effectiveness analysis was applied to the soybean (Glycine max L. (Merrill)). Flood-tolerant mutant and abscisic acid (ABA)-treated soybean plants were used as the flood-tolerant materials. Among the primary metabolism, glycolysis, fermentation, and tricarboxylic acid cycle were markedly affected under flooding. Fifteen proteins, which were related to the affected processes, displayed similar protein profiles in the mutant and ABA-treated soybean plants. Protein levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aconitase 1, and 2-oxoglutarate dehydrogenase were higher in flood-tolerant materials than in wild-type soybean plants under flood conditions. These three proteins were positioned in each of the three enzyme groups revealed by our computational genetic modification effectiveness analysis, and the three proteins configured a candidate set of genes to promote flood tolerance. Additionally, transcript levels of GAPDH were similar in flood-tolerant materials and in unstressed plants. These results suggest that proteins related to energy metabolism might play an essential role to confer flood tolerance in soybeans.

Background Bacillus amyloliquefaciens is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. B.amyloliquefaciens LB1ba02 is a production strain suitable for secreting mesophilic α-amylase in the industry. Nevertheless, due to the low transformation efficiency and restriction-modification system, the development of its CRISPR tool lags far behind other species and strains from the genus Bacillus . This work was undertaken to develop a fast and efficient gene-editing tool in B.amyloliquefaciens LB1ba02. Results In this study, we fused the nuclease-deficient mutant Cas9n (D10A) of Cas9 with activation-induced cytidine deaminase (AID) and developed a fast and efficient base editing system for the first time in B. amyloliquefaciens LB1ba02. The system was verified by inactivating the pyrF gene coding orotidine 5'-phosphate decarboxylase and the mutant could grow normally on M9 medium supplemented with 5-fluoroorotic acid (5-FOA) and uridine (U). Our base editing system has a 6nt editing window consisting of an all-in-one temperature-sensitive plasmid that facilitates multiple rounds of genome engineering in B. amyloliquefaciens LB1ba02. The total editing efficiency of this method reached 100% and it achieved simultaneous editing of three loci with an efficiency of 53.3%. In addition, based on the base editing CRISPR/Cas9n-AID system, we also developed a single plasmid CRISPR/Cas9n system suitable for rapid gene knockout and integration. The knockout efficiency for a single gene reached 93%. Finally, we generated 4 genes ( aprE , nprE , wprA , and bamHIR ) mutant strain, LB1ba02△4. The mutant strain secreted 1.25-fold more α-amylase into the medium than the wild-type strain. Conclusions The CRISPR/Cas9n-AID and CRISPR/Cas9n systems developed in this work proved to be a fast and efficient genetic manipulation tool in a restriction-modification system and poorly transformable strain.