CRISPR-Cas engineering in food science and sustainable agriculture: recent advancements and applications.

The developments in the food supply chain to support the growing population of the world is one of today's most pressing issues, and to achieve this goal improvements should be performed in both crops and microbes. For this purpose, novel approaches such as genome editing (GE) methods have upgraded the biological sciences for genome manipulation and, among such methods, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are the main exciting innovations since the Green Revolution. CRISPR/Cas systems can be a potent tool for the food industry, improvement of agricultural crops and even for protecting food-grade bacteria from foreign genetic invasive elements. This review introduces the history and mechanism of the CRISPR-Cas system as a genome editing tool and its applications in the vaccination of starter cultures, production of antimicrobials and bioactive compounds, and genome editing of microorganisms.

Antimicrobial peptides used as growth promoters in livestock production.

Antibiotic growth promoters (AGPs) have been administered in livestock for decades to improve food digestion in growing animals, while also contributing to the control of microbial pathogens. The long-term and indiscrimate use of AGPs has generated genetic modifications in bacteria, leading to antimicrobial resistance (AMR), which can be disseminated to commensal and pathogenic bacteria. Thus, antimicrobial peptides (AMPs) are used to replaced AGPs. AMPs are found in all domains of life, and their cationic characteristics can establish electrostatic interactions with the bacterial membrane. These molecules used as growth promoters can present benefits for nutrient digestibility, intestinal microbiota, intestinal morphology, and immune function activities. Therefore, this review focuses on the application of AMPs with growth promoting potential in livestock, as an alternative to conventional antibiotic growth promoters, in an attempt to control AMR. KEY POINTS: • The long-term and indiscriminate use of AGPs in animal food can cause AMR. • AMPs can be used as substitute of antibiotics in animal food suplementation. • Animal food suplementated with AMPs can provied economic efficiency and sustainable livestock production.

Quantitative expression of microRNAs in Brassica oleracea infected with Xanthomonas campestris pv. campestris.

Brassica oleracea var. capitata (cabbage) is an economically important crop affected by black rot disease caused by Xanthomonas campestris pv. campestris (Xcc). MicroRNAs (miRNAs) play an important role in plant defense modulation and therefore the analysis of these molecules can help better understand plant-pathogen interactions. In this study, we report the differential expression of four miRNAs that seem to participate in the plant response to Xcc infection. Northern Blot and RT-qPCR techniques were used to measure miRNA expression in resistant (União) and susceptible (Kenzan) cultivars. From 6 miRNAs analyzed, 4 were detected and differentially expressed, showing a down- and upregulated expression profile in susceptible and resistant cultivars, respectively. These results suggest that miR156, miR167, miR169, and miR390 could play a role in B. oleracea resistance enhancement against Xcc and could be explored as potential resistance markers in B. oleracea-Xcc interaction.

Cloning and characterization of novel cyclotides genes from South American plants.

Cyclotides are multifunctional plant cyclic peptides containing 28-37 amino acid residues and a pattern of three disulfide bridges, forming a motif known as the cyclic cystine knot. Due to their high biotechnological potential, the sequencing and characterization of cyclotide genes are crucial not only for cloning and establishing heterologous expression strategies, but also to understand local plant evolution in the context of host-pathogen relationships. Here, two species from the Brazilian Cerrado, Palicourea rigida (Rubiaceae) and Pombalia lanata (A.St.-Hil.) Paula-Souza (Violaceae), were used for cloning and characterizing novel cyclotide genes. Using 3' and 5' RACE PCR and sequencing, two full cDNAs, named parigidin-br2 (P. rigida) and hyla-br1 (P. lanata), were isolated and shown to have similar genetic structures to other cyclotides. Both contained the conserved ER-signal domain, N-terminal prodomain, mature cyclotide domain and a C-terminal region. Genomic sequencing of parigidin-br2 revealed two different gene copies: one intronless allele and one presenting a rare 131-bp intron. In contrast, genomic sequencing of hyla-br1 revealed an intronless gene-a common characteristic of members of the Violaceae family. Parigidin-br2 5' and 3' UTRs showed the presence of 12 putative candidate sites for binding of regulatory proteins, suggesting that the flanking and intronic regions of the parigidin-br2 gene must play important roles in transcriptional rates and in the regulation of temporal and spatial gene expression. The high degree of genetic similarity and structural organization among the cyclotide genes isolated in the present study from the Brazilian Cerrado and other well-characterized plant cyclotides may contribute to a better understanding of cyclotide evolution.

Antiviral Activities of Mastoparan-L-Derived Peptides against Human Alphaherpesvirus 1.

Human alphaherpesvirus 1 (HSV-1) is a significantly widespread viral pathogen causing recurrent infections that are currently incurable despite available treatment protocols. Studies have highlighted the potential of antimicrobial peptides sourced from Vespula lewisii venom, particularly those belonging to the mastoparan family, as effective against HSV-1. This study aimed to demonstrate the antiviral properties of mastoparans, including mastoparan-L [I5, R8], mastoparan-MO, and [I5, R8] mastoparan, against HSV-1. Initially, Vero cell viability was assessed in the presence of these peptides, followed by the determination of antiviral activity, mechanism of action, and dose-response curves through plaque assays. Structural analyses via circular dichroism and nuclear magnetic resonance were conducted, along with evaluating membrane fluidity changes induced by [I5, R8] mastoparan using fluorescence-labeled lipid vesicles. Cytotoxic assays revealed high cell viability (>80%) at concentrations of 200 µg/mL for mastoparan-L and mastoparan-MO and 50 µg/mL for [I5, R8] mastoparan. Mastoparan-MO and [I5, R8] mastoparan exhibited over 80% HSV-1 inhibition, with up to 99% viral replication inhibition, particularly in the early infection stages. Structural analysis indicated an α-helical structure for [I5, R8] mastoparan, suggesting effective viral particle disruption before cell attachment. Mastoparans present promising prospects for HSV-1 infection control, although further investigation into their mechanisms is warranted.

Anti-Staphy Peptides Rationally Designed from Cry10Aa Bacterial Protein.

Bacterial infections pose a significant threat to human health, constituting a major challenge for healthcare systems. Antibiotic resistance is particularly concerning in the context of treating staphylococcal infections. In addressing this challenge, antimicrobial peptides (AMPs), characterized by their hydrophobic and cationic properties, unique mechanism of action, and remarkable bactericidal and immunomodulatory capabilities, emerge as promising alternatives to conventional antibiotics for tackling bacterial multidrug resistance. This study focuses on the Cry10Aa protein as a template for generating AMPs due to its membrane-penetrating ability. Leveraging the Joker algorithm, six peptide variants were derived from α-helix 3 of Cry10Aa, known for its interaction with lipid bilayers. In vitro, antimicrobial assays determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) required for inhibiting the growth of Staphylococcus aureus, Escherichia coli, Acinetobacter baummanii, Enterobacter cloacae, Enterococcus facallis, Klebsiella pneumonia, and Pseudomonas aeruginosa. Time-kill kinetics were performed using the parental peptide AMPCry10Aa, as well as AMPCry10Aa_1 and AMPCry10Aa_5, against E. coli ATCC, S. aureus 111 and S. aureus ATCC strains showing that AMPCry10Aa_1 and AMPCry10Aa_5 peptides can completely reduce the initial bacterial load with less than 2 h of incubation. AMPCry10Aa_1 and AMPCry 10Aa_5 present stability in human serum and activity maintenance up to 37 °C. Cytotoxicity assays, conducted using the MTT method, revealed that all of the tested peptides exhibited cell viability >50% (IC50). The study also encompassed evaluations of the structure and physical-chemical properties. The three-dimensional structures of AMPCry10Aa and AMPCry10Aa_5 were determined through nuclear magnetic resonance (NMR) spectroscopy, indicating the adoption of α-helical segments. Electron paramagnetic resonance (EPR) spectroscopy elucidated the mechanism of action, demonstrating that AMPCry10Aa_5 enters the outer membranes of E. coli and S. aureus, causing substantial increases in lipid fluidity, while AMPCry10Aa slightly increases lipid fluidity in E. coli. In conclusion, the results obtained underscore the potential of Cry10Aa as a source for developing antimicrobial peptides as alternatives to conventional antibiotics, offering a promising avenue in the battle against antibiotic resistance.

Employment of the CRISPR/Cas9 system to improve cellulase production in Trichoderma reesei.

Trichoderma reesei has been explored intensively in the laboratory and on an industrial scale for its highly potent cellulase secretion machinery since its characterization over 70 years ago. Emergence of new genetic tools over the past decade has strengthened the understanding of mechanism involved in transcription of cellulase genes in fungi and provided a boost to edit them at molecular level. Since several transcriptional factors work synergistically for cellulase expression in fungi; engineering of cellulase secretome for enhanced cellulase titer require combined manipulation of these factors. In the same context, CRISPR/Cas9 has emerged as a powerful, versatile genetic engineering tool for multiplex gene editing in fungi. It is true that considerable efforts with CRISPR technologies have largely developed fungal genetic engineering, but its application in fungi is still challenging and limited. The present review illustrates the precision, strengths and challenges of using CRISPR/Cas9 technology for cellulase engineering in T. reesei, highlighting key strategies that could be employed for strain improvement.

Nanoparticles in association with antimicrobial peptides (NanoAMPs) as a promising combination for agriculture development.

Antimicrobial peptides are small molecules, up to 10 kDa, present in all kingdoms of life, including in plants. Several studies report that these molecules have a broad spectrum of activity, including antibacterial, antifungal, antiviral, and insecticidal activity. Thus, they can be employed in agriculture as alternative tools for phytopathogen and pest control. However, the application of peptides in agriculture can present challenges, such as loss of activity due to degradation of these molecules, off-target effects, and others. In this context, nanotechnology can offer versatile structures, including metallic nanoparticles, liposomes, polymeric nanoparticles, nanofibers, and others, which might act both in protection and in release of AMPs. Several polymers and biomaterials can be employed for the development of nanostructures, such as inorganic metals, natural or synthetic lipids, synthetic and hybrid polymers, and others. This review addresses the versatility of NanoAMPs (Nanoparticles in association with antimicrobial peptides), and their potential applications in agribusiness, as an alternative for the control of phytopathogens in crops.

Priming of defense-related genes in Brassica oleracea var. capitata using concentrated metabolites produced by Rhizobium tropici CIAT 899.

To verify the potential of metabolites extracted from Rhizobium tropici to trigger the priming of defense responses in cruciferous plants, we analyzed the expression of defense-related genes by qRT-PCR. Brassica oleracea var. capitata, susceptible to Xanthomonas campestris pv. campestris, were grown in greenhouse conditions. At 18 days after sowing, plants were inoculated with 1 mL of 1% concentrated metabolites produced by R. tropici (CM-RT) in the root. In a second experiment, leaves were sprayed with 1 mL of a solution containing 1% CM-RT. Aerial and root tissue were collected separately at 0 (non-treated control condition), 24, and 48 h after application, submitted to RNA extraction and gene expression analysis by qRT-PCR. The results showed that, after root treatment with CM-RT, most evaluated genes were upregulated at 24 h after application and downregulated at 48 h after application in roots, while in leaves, genes were downregulated both at 24 and 48 h after application. On the other hand, leaf treatment with CM-RT showed that most evaluated genes in leaves and roots were upregulated at 24 and 48 h after application. These results indicate that the effect of CM-RT applied in roots seems restricted to the applied region and is not sustained, while the application in leaves results in a more systemic response and maintenance of the effect of CM-RT for a longer period. The results obtained in this study emphasize the biotechnological potential of using metabolites of R. tropici as an elicitor of active defense responses in plants.

Screening for cysteine-stabilized scaffolds for developing proteolytic-resistant AMPs.

Antimicrobial peptides (AMP) are present in all organisms and can present several activities and potential applications in human and animal health. Screening these molecules scaffolds represents a key point for discovering and developing novel biotechnological products, including antimicrobial, antiviral and anticancer drugs candidates and insecticidal molecules with potential applications in agriculture. Therefore, considering the amount of biological data currently deposited on public databases, computational approaches have been commonly used to predicted and identify novel cysteine-rich peptides scaffolds with known or unknown biological properties. Here, we describe a step-by-step in silico screening for cysteine-rich peptides employing molecular modeling (with a core focus on comparative modeling) and atomistic molecular dynamics simulations. Moreover, we also present the concept of additional tools aiming at the computer-aided screening of new Cs-AMPs based drug candidates. After the computational screening and peptide chemical synthesis, we also provide the reader with a step-by-step in vitro activity evaluation of these candidates, including antibacterial, antifungal, and antiviral assays.

Biotechnological applications of versatile plant lipid transfer proteins (LTPs).

Plant AMPs are usually cysteine-rich, and can be classified in several classes, including lipid transfer proteins (LTPs). LTPs are small plant cationic peptides, and can be classified in two subclasses, LTP1 (9-10 kDa) and LTP2 (7 kDa). They have been identified and isolated from various plant species and can be involved in a number of processes, including responses against several phytopathogens. LTP1 presents 4 parallel α- helices and a 310-helix fragment. These structures form a tunnel with large and small entrances. LTP2 presents 3 parallel α- helices, which form a cavity with triangular structure. Both LTP subclasses present a hydrophobic cavity, which makes interaction with different lipids and general hydrophobic molecules possible. Several studies report a broad spectrum of activity of plant LTPs, including antibacterial, antifungal, antiviral, antitumoral, and insecticidal activity. Thus, these molecules can be employed in human and animal health as an alternative to the conventional treatment of disease, well as providing the source of novel drugs. However, employing peptides in human health can present challenges, such as the toxicity of peptides, the difference between the results found in in vitro assays and in pre-clinical or clinical tests and their low efficiency against Gram-negative bacteria. In this context, plant LTPs can be an interesting alternative means by which to bypass such challenges. This review addresses the versatility of plant LTPs, their broad spectrum of activities and their potential applications in human and animal health and in agricultural production, and examines challenges in their biotechnological application.

CRISPR Genome Editing Technology: A Powerful Tool Applied to Developing Agribusiness.

The natural increase of the world's population implies boosting agricultural demand. In the current non-optimistic global scenario, where adverse climate changes come associated with substantial population growth, the main challenge in agribusiness is food security. Recently, the CRISPR/Cas system has emerged as a friendly gene editing biotechnological tool, enabling a precise manipulation of genomes and enhancement of desirable traits in several organisms. This review highlights the CRISPR/Cas system as a paramount tool for the improvement of agribusiness products and brings up-to-date findings showing its potential applications in improving agricultural-related traits in major plant crops and farm animals, all representing economic-relevant commodities responsible for feeding the world. Several applied pieces of research have successfully demonstrated the CRISPR/Cas ability in boosting interesting traits in agribusiness products, including animal productivity and welfare, crop yield growth, and seed quality, reflecting positive impacts in both socioeconomics and human health aspects. Hence, the CRISPR/Cas system has revolutionized bioscience and biotechnology, and its concrete application in agribusiness goods is on the horizon.

Remodeling of the cell wall as a drought-tolerance mechanism of a soybean genotype revealed by global gene expression analysis.

Drought stress is major abiotic stress that affects soybean production. Therefore, it is widely desirable that soybean becomes more tolerant to stress. To provide insights into regulatory mechanisms of the stress response, we compared the global gene expression profiles from leaves of two soybean genotypes that display different responses to water-deficit (BR 16 and Embrapa 48, drought-sensitive and drought-tolerant, respectively). After the RNA-seq analysis, a total of 5335 down-regulated and 3170 up-regulated genes were identified in the BR16. On the other hand, the number of genes differentially expressed was markedly lower in the Embrapa 48, 355 up-regulated and 471 down-regulated genes. However, induction and expression of protein kinases and transcription factors indicated signaling cascades involved in the drought tolerance. Overall, the results suggest that the metabolism of pectin is differently modulated in response to drought stress and may play a role in the soybean defense mechanism against drought. This occurs via an increase of the cell wall plasticity and crosslink, which contributed to a higher hydraulic conductance (K f) and relative water content (RWC%). The drought-tolerance mechanism of the Embrapa 48 genotype involves remodeling of the cell wall and increase of the hydraulic conductance to the maintenance of cell turgor and metabolic processes, resulting in the highest leaf RWC, photosynthetic rate (A), transpiration (E) and carboxylation (A/C i). Thus, we concluded that the cell wall adjustment under drought is important for a more efficient water use which promoted a more active photosynthetic metabolism, maintaining higher plant growth under drought stress. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-021-00043-4.

Genotype-dependent changes of gene expression during somatic embryogenesis in oil palm hybrids (Elaeis oleifera x E. guineensis).

To understand the molecular processes triggered during the different steps of somatic embryogenesis (SE) in oil palm, the expression of 19 genes associated to SE identified in proteomic and transcriptomic studies was investigated by qRT-PCR. To evaluate the differential expression of these genes, two interspecific hybrid genotypes (Elaeis oleifera x Elaeis guineensis) contrasting for the acquisition of embryogenic competence were used. Aclorophyllated leaves of both hybrids, one responsive (B351733) and the other non-responsive (B352933) to SE were submitted to callus induction and collected at different time points: 0 (before induction), 14, 30, 90 and 150 days of callus induction (doi). The results obtained showed that all evaluated genes were downregulated at 14 doi in the responsive genotype when compared to the non-responsive. It was also possible to observe that most of the genes changed their expression behavior at 30 doi and were upregulated thereafter until 150 doi, with the exception of the pathogenesis-related PRB1-3-like (PRB1-3) gene, which did not show differential expression at 30 doi and was downregulated at 90 and 150 doi when compared to the non-responsive hybrid. These results indicate that 30 doi is a turning point in gene expression, probably associated to embryogenic competence acquisition. We also show that the expression behavior of the responsive genotype is more stable than that of the non-responsive when the different induction time points are compared to 0 doi (before induction). Moreover, the results obtained in this study corroborate our hypothesis that the regulation of genes involved in the control of oxidative stress and energy metabolism are crucial for the acquisition of embryogenic competence in oil palm.

Validation of an in vitro system for studies of pathogenicity mechanisms in Xanthomonas campestris.

Several minimal media capable of inducing pathogenicity genes have been used to study plant-pathogen interactions. An in planta assay to study a closer interaction between the bacteria and the host was also developed and has been employed by our group. In order to determine whether growth medium could be improved to better approximate in planta conditions beyond that offered by the defined minimal medium XVM1, we compared the expression of 20 Xanthomonas campestris pv. campestris (Xcc) genes by quantitative reverse transcription - polymerase chain reaction (qRT-PCR) under in vivo (bacteria recovered from the plant) and in vitro (rich medium NYG, minimal medium XVM1 and XVM1 + leaf extract) growth systems. The results showed a higher expression level of the genes in the in planta system when compared to growth in culture media. In planta growth is closest to a real interaction condition and captures the complexity of the plant cell environment; however, this system has some limitations. The main finding of our work is that the addition of plant extract to XVM1 medium results in a gene expression profile that better matches the in planta profile, when compared with the XVM1 medium alone, giving support to the use of plant extract to study pathogenicity mechanisms in Xanthomonas.