The SARS-CoV-2 Spike Protein Receptor-Binding Domain Expressed in Rice Callus Features a Homogeneous Mix of Complex-Type Glycans.

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 is required for the infection of human cells. It is the main target that elicits neutralizing antibodies and also a major component of diagnostic kits. The large demand for this protein has led to the use of plants as a production platform. However, it is necessary to determine the N-glycan structures of an RBD to investigate its efficacy and functionality as a vaccine candidate or diagnostic reagent. Here, we analyzed the N-glycan profile of the RBD produced in rice callus. Of the two potential N-glycan acceptor sites, we found that one was not utilized and the other contained a mixture of complex-type N-glycans. This differs from the heterogeneous mixture of N-glycans found when an RBD is expressed in other hosts, including Nicotiana benthamiana. By comparing the glycosylation profiles of different hosts, we can select platforms that produce RBDs with the most beneficial N-glycan structures for different applications.

Cyanovirin-N binds to select SARS-CoV-2 spike oligosaccharides outside of the receptor binding domain and blocks infection by SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped positive stranded RNA virus which has caused the recent deadly pandemic called COVID-19. The SARS-CoV-2 virion is coated with a heavily glycosylated Spike glycoprotein which is responsible for attachment and entry into target cells. One, as yet unexploited strategy for preventing SARS-CoV-2 infections, is the targeting of the glycans on Spike. Lectins are carbohydrate-binding proteins produced by plants, algae, and cyanobacteria. Some lectins can neutralize enveloped viruses displaying external glycoproteins, offering an alternative therapeutic approach for the prevention of infection with virulent ß-coronaviruses, such as SARS-CoV-2. Here we show that the cyanobacterial lectin cyanovirin-N (CV-N) can selectively target SARS-CoV-2 Spike oligosaccharides and inhibit SARS-CoV-2 infection in vitro and in vivo. CV-N neutralizes Delta and Omicron variants in vitro better than earlier circulating viral variants. CV-N binds selectively to Spike with a Kd as low as 15 nM and a stoichiometry of 2 CV-N: 1 Spike but does not bind to the receptor binding domain (RBD). Further mapping of CV-N binding sites on Spike shows that select high-mannose oligosaccharides in the S1 domain of Spike are targeted by CV-N. CV-N also reduced viral loads in the nares and lungs in vivo to protect hamsters against a lethal viral challenge. In summary, we present an anti-coronavirus agent that works by an unexploited mechanism and prevents infection by a broad range of SARS-CoV-2 strains.

Physicochemical characterization of the recombinant lectin scytovirin and microbicidal activity of the SD1 domain produced in rice against HIV-1.

KEY MESSAGE: Rice-produced SD1 retains its physicochemical properties and provides efficient pre-exposure HIV-1 prophylaxis against infection in vitro. Scytovirin (SVN) is an HIV-neutralizing lectin that features two structural domains (SD1 and SD2) that bind to HIV-1 envelope glycoproteins. We expressed SD1 in rice seeds as a potential large-scale production platform and confirmed that rice-derived SD1 binds the HIV-1 envelope glycoprotein gp120 in vitro. We analyzed the thermodynamic properties of SD1 compared to full-size SVN (produced in E. coli) by isothermal titration and differential scanning calorimetry to characterize the specific interactions between SVN/SD1 and gp120 as well as to high-mannose oligosaccharides. SVN bound with moderate affinity (Kd = 1.5 µM) to recombinant gp120, with 2.5-fold weaker affinity to nonamannoside (Kd of 3.9 µM), and with tenfold weaker affinity to tetramannoside (13.8 µM). The melting temperature (Tm) of full-size SVN was 59.1 °C and the enthalpy of unfolding (ΔHunf) was 16.4 kcal/mol, but the Tm fell when SVN bound to nonamannoside (56.5 °C) and twice as much energy was required for unfolding (ΔHunf = 33.5 kcal/mol). Interestingly, binding to tetramannoside destabilized the structure of SD1 (ΔTm ~ 11.5 °C) and doubled the enthalpy of unfolding, suggesting a dimerization event. The similar melting phenomenon shared by SVN and SD1 in the presence of oligomannose confirmed their conserved oligosaccharide-binding mechanisms. SD1 expressed in transgenic rice was able to neutralize HIV-1 in vitro. SD1 expressed in rice, therefore, is suitable as a microbicide component.

Genome editing in cereal crops: an overview.

Genome-editing technologies offer unprecedented opportunities for crop improvement with superior precision and speed. This review presents an analysis of the current state of genome editing in the major cereal crops- rice, maize, wheat and barley. Genome editing has been used to achieve important agronomic and quality traits in cereals. These include adaptive traits to mitigate the effects of climate change, tolerance to biotic stresses, higher yields, more optimal plant architecture, improved grain quality and nutritional content, and safer products. Not all traits can be achieved through genome editing, and several technical and regulatory challenges need to be overcome for the technology to realize its full potential. Genome editing, however, has already revolutionized cereal crop improvement and is poised to shape future agricultural practices in conjunction with other breeding innovations.

Contributions of the international plant science community to the fight against infectious diseases in humans-part 2: Affordable drugs in edible plants for endemic and re-emerging diseases.

The fight against infectious diseases often focuses on epidemics and pandemics, which demand urgent resources and command attention from the health authorities and media. However, the vast majority of deaths caused by infectious diseases occur in endemic zones, particularly in developing countries, placing a disproportionate burden on underfunded health systems and often requiring international interventions. The provision of vaccines and other biologics is hampered not only by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, but also by challenges caused by distribution and storage, particularly in regions without a complete cold chain. In this review article, we consider the potential of molecular farming to address the challenges of endemic and re-emerging diseases, focusing on edible plants for the development of oral drugs. Key recent developments in this field include successful clinical trials based on orally delivered dried leaves of Artemisia annua against malarial parasite strains resistant to artemisinin combination therapy, the ability to produce clinical-grade protein drugs in leaves to treat infectious diseases and the long-term storage of protein drugs in dried leaves at ambient temperatures. Recent FDA approval of the first orally delivered protein drug encapsulated in plant cells to treat peanut allergy has opened the door for the development of affordable oral drugs that can be manufactured and distributed in remote areas without cold storage infrastructure and that eliminate the need for expensive purification steps and sterile delivery by injection.

Contributions of the international plant science community to the fight against human infectious diseases - part 1: epidemic and pandemic diseases.

Infectious diseases, also known as transmissible or communicable diseases, are caused by pathogens or parasites that spread in communities by direct contact with infected individuals or contaminated materials, through droplets and aerosols, or via vectors such as insects. Such diseases cause ˜17% of all human deaths and their management and control places an immense burden on healthcare systems worldwide. Traditional approaches for the prevention and control of infectious diseases include vaccination programmes, hygiene measures and drugs that suppress the pathogen, treat the disease symptoms or attenuate aggressive reactions of the host immune system. The provision of vaccines and biologic drugs such as antibodies is hampered by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, particularly in developing countries where infectious diseases are prevalent and poorly controlled. Molecular farming, which uses plants for protein expression, is a promising strategy to address the drawbacks of current manufacturing platforms. In this review article, we consider the potential of molecular farming to address healthcare demands for the most prevalent and important epidemic and pandemic diseases, focussing on recent outbreaks of high-mortality coronavirus infections and diseases that disproportionately affect the developing world.

Potential Applications of Plant Biotechnology against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus responsible for an ongoing human pandemic (COVID-19). There is a massive international effort underway to develop diagnostic reagents, vaccines, and antiviral drugs in a bid to slow down the spread of the disease and save lives. One part of that international effort involves the research community working with plants, bringing researchers from all over the world together with commercial enterprises to achieve the rapid supply of protein antigens and antibodies for diagnostic kits, and scalable production systems for the emergency manufacturing of vaccines and antiviral drugs. Here, we look at some of the ways in which plants can and are being used in the fight against COVID-19.

Applications of multiplex genome editing in higher plants.

Multiplex genome editing involves the simultaneous targeting of multiple related or unrelated targets. The latter is most straightforward using the CRISPR/Cas9 system because multiple gRNAs can be delivered either as independent expression cassettes with their own promoters or as polycistronic transcripts processed into mature gRNAs by endogenous or introduced nucleases. Multiplex genome editing in plants initially focused on input traits such as herbicide resistance, but has recently expanded to include hormone biosynthesis and perception, metabolic engineering, plant development and molecular farming, with more than 100 simultaneous targeting events reported. Usually the coding region is targeted but recent examples also include promoter modifications to generate mutants with varying levels of gene expression.