Genome editing for resistance against plant pests and pathogens.

The conventional breeding of crops struggles to keep up with increasing food needs and ever-adapting pests and pathogens. Global climate changes have imposed another layer of complexity to biological systems, increasing the challenge to obtain improved crop cultivars. These dictate the development and application of novel technologies, like genome editing (GE), that assist targeted and fast breeding programs in crops, with enhanced resistance to pests and pathogens. GE does not require crossings, hence avoiding the introduction of undesirable traits through linkage in elite varieties, speeding up the whole breeding process. Additionally, GE technologies can improve plant protection by directly targeting plant susceptibility (S) genes or virulence factors of pests and pathogens, either through the direct edition of the pest genome or by adding the GE machinery to the plant genome or to microorganisms functioning as biocontrol agents (BCAs). Over the years, GE technology has been continuously evolving and more so with the development of CRISPR/Cas. Here we review the latest advancements of GE to improve plant protection, focusing on CRISPR/Cas-based genome edition of crops and pests and pathogens. We discuss how other technologies, such as host-induced gene silencing (HIGS) and the use of BCAs could benefit from CRISPR/Cas to accelerate the development of green strategies to promote a sustainable agriculture in the future.

Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody.

Amplification and overexpression of HER2 (human epidermal growth factor receptor 2), an ErbB2 receptor tyrosine kinase, have been implicated in human cancer and metastasis. A bispecific tetravalent anti-HER2 antibody (anti-HER2-Bs), targeting two non-overlapping epitopes on HER2 in domain IV (trastuzumab) and domain II (39S), has been reported to induce rapid internalization and efficient degradation of HER2 receptors. In this study, we investigated the molecular mechanism of this antibody-induced rapid HER2 internalization and intracellular trafficking. Using quantitative fluorescent imaging, we compared the internalization kinetics of anti-HER2-Bs and its parental arm antibodies, alone or in combinations and under various internalization-promoting conditions. The results demonstrated that concurrent engagement of both epitopes was necessary for rapid anti-HER2-Bs internalization. Cellular uptake of anti-HER2-Bs and parental arm antibodies occurred via clathrin-dependent endocytosis; however, inside the cells antibodies directed different trafficking pathways. Trastuzumab dissociated from HER2 in 2 h, enabling the receptor to recycle, whereas anti-HER2-Bs stayed associated with the receptor throughout the entire endocytic pathway, promoting receptor ubiquitination, trafficking to the lysosomes, and efficient degradation. Consistent with routing HER2 to degradation, anti-HER2-Bs significantly reduced HER2 shedding and altered its exosomal export. Collectively, these results enable a better understanding of the mechanism of action of anti-Her2-Bs and can guide the rational design of anti-HER2 therapeutics as well as other bispecific molecules.

Rotavirus vaccine efficacy: current status and areas for improvement.

The difference noted in Rotavirus vaccine efficiency between high and low income countries correlates with the lack of universal access to clean water and higher standards of hygiene. Overcoming these obstacles will require great investment and also time, therefore more effective vaccines should be developed to meet the needs of those who would benefit the most from them. Increasing our current knowledge of mucosal immunity, response to Rotavirus infection and its modulation by circadian rhythms could point at actionable pathways to improve vaccination efficacy, especially in the case of individuals affected by environmental enteropathy. Also, a better understanding and validation of Rotavirus entry factors as well as the systematic monitoring of dominant strains could assist in tailoring vaccines to individual's needs. Another aspect that could improve vaccine efficiency is targeting to M cells, for which new ligands could potentially be sought. Finally, alternative mucosal adjuvants and vaccine expression, storage and delivery systems could have a positive impact in the outcome of Rotavirus vaccination.

Anti-ICOSL New Antigen Receptor Domains Inhibit T Cell Proliferation and Reduce the Development of Inflammation in the Collagen-Induced Mouse Model of Rheumatoid Arthritis.

Lymphocyte costimulation plays a central role in immunology, inflammation, and immunotherapy. The inducible T cell costimulator (ICOS) is expressed on T cells following peptide: MHC engagement with CD28 costimulation. The interaction of ICOS with its sole ligand, the inducible T cell costimulatory ligand (ICOSL; also known as B7-related protein-1), triggers a number of key activities of T cells including differentiation and cytokine production. Suppression of T cell activation can be achieved by blocking this interaction and has been shown to be an effective means of ameliorating disease in models of autoimmunity. In this study, we isolated specific anti-ICOSL new antigen receptor domains from a synthetic phage display library and demonstrated their ability to block the ICOS/ICOSL interaction and inhibit T cell proliferation. Anti-mouse ICOSL domains, considered here as surrogates for the use of anti-human ICOSL domains in patient therapy, were tested for efficacy in a collagen-induced mouse model of rheumatoid arthritis where they significantly decreased the inflammation of joints and delayed and reduced overall disease progression and severity.

In Vitro Maturation of a Humanized Shark VNAR Domain to Improve Its Biophysical Properties to Facilitate Clinical Development.

Molecular engineering to increase the percentage identity to common human immunoglobulin sequences of non-human therapeutic antibodies and scaffolds has become standard practice. This strategy is often used to reduce undesirable immunogenic responses, accelerating the clinical development of candidate domains. The first humanized shark variable domain (VNAR) was reported by Kovalenko and colleagues and used the anti-human serum albumin (HSA) domain, clone E06, as a model to construct a number of humanized versions including huE06v1.10. This study extends this work by using huE06v1.10 as a template to isolate domains with improved biophysical properties and reduced antigenicity. Random mutagenesis was conducted on huE06v1.10 followed by refinement of clones through an off-rate ranking-based selection on target antigen. Many of these next-generation binders retained high affinity for target, together with good species cross-reactivity. Lead domains were assessed for any tendency to dimerize, tolerance to N- and C-terminal fusions, affinity, stability, and relative antigenicity in human dendritic cell assays. Functionality of candidate clones was verified in vivo through the extension of serum half-life in a typical drug format. From these analyses the domain, BA11, exhibited negligible antigenicity, high stability and high affinity for mouse, rat, and HSA. When these attributes were combined with demonstrable functionality in a rat model of PK, the BA11 clone was established as our clinical candidate.

The geminivirus nuclear shuttle protein NSP inhibits the activity of AtNSI, a vascular-expressed Arabidopsis acetyltransferase regulated with the sink-to-source transition.

DNA viruses can suppress or enhance the activity of cellular acetyltransferases to regulate virus gene expression and to affect cell cycle progression in support of virus replication. A role for protein acetylation in regulating the nuclear export of the bipartite geminivirus (Begomovirus) DNA genome was recently suggested by the findings that the viral movement protein NSP, a nuclear shuttle protein, interacts with the Arabidopsis (Arabidopsis thaliana) nuclear acetyltransferase AtNSI (nuclear shuttle protein interactor), and that this interaction and NSI expression are necessary for cabbage leaf curl virus infection and pathogenicity. To further investigate the consequences of NSI-NSP interactions, and the potential role of NSI in Arabidopsis growth and development, we used a reverse yeast two-hybrid selection and deletion analysis to identify NSI mutants that failed to interact with NSP, and promoter fusions to a uidA reporter gene to analyze the pattern of NSI expression during plant development. We found that NSI self assembles into highly active enzyme complexes and that high concentrations of NSP, in the absence of viral DNA, can inhibit NSI activity in vitro. Based on our detailed analysis of three NSI missense mutants, we identified an 88-amino acid putative domain, which spans NSI residues 107 to 194, as being required for both NSI oligomerization and its interaction with NSP. Finally, we found that NSI is predominantly transcribed in vascular cells, and that its expression is developmentally regulated in a manner that resembles the sink-to-source transition. Our data indicate that NSP can inhibit NSI activity by interfering with its assembly into highly active complexes, and suggest a mechanism by which NSP can both recruit NSI to regulate nuclear export of the viral genome and down-regulate NSI activity on cellular targets, perhaps to affect cellular differentiation and favor virus replication.

Interaction of the movement protein NSP and the Arabidopsis acetyltransferase AtNSI is necessary for Cabbage leaf curl geminivirus infection and pathogenicity.

DNA viruses can modulate the activity of cellular acetyltransferases to regulate virus gene expression and to affect cell cycle progression in order to support virus replication. A role for protein acetylation in regulating the nuclear export of the bipartite geminivirus DNA genome was recently suggested by the findings that the viral movement protein NSP, which shuttles the viral genome between the nucleus and the cytoplasm, interacts with a novel Arabidopsis acetyltransferase, AtNSI, and the increased expression of AtNSI enhances susceptibility to Cabbage leaf curl virus infection. To further investigate the interaction of NSP and AtNSI and to establish the importance of this interaction in virus infections, we used a reverse yeast two-hybrid selection and deletion analysis to identify NSP mutants that were impaired in their ability to bind AtNSI. These mutants identified a 38-amino-acid region of NSP, to which no function had so far been assigned, as being necessary for NSP-AtNSI interaction. Three NSP missense mutants were analyzed in detail and were found to be comparable to wild-type NSP in their levels of accumulation, nucleocytoplasmic shuttling, DNA binding, and cooperative interaction with the viral cell-to-cell movement protein MP. Despite this, Cabbage leaf curl virus that expressed each mutated NSP was defective in its ability to infect Arabidopsis, exhibiting lower levels of infectivity than the wild-type virus, and delayed systemic spread of the virus and attenuated disease symptoms. Our data demonstrate the importance of the interaction of NSP with AtNSI for virus infection and pathogenicity.

A novel Arabidopsis acetyltransferase interacts with the geminivirus movement protein NSP.

Protein acetylation is important in regulating DNA-templated processes specifically and protein-protein interactions more generally in eukaryotes. The geminivirus movement protein NSP is essential for virus movement, shuttling the viral DNA genome between the nucleus and the cytoplasm. We have identified a novel Arabidopsis protein, AtNSI, that interacts with NSP. AtNSI is highly conserved among widely divergent plants. Biochemical studies show that its interaction with NSP is direct and that AtNSI acetylates histones, but not NSP, in vitro. Rather, AtNSI specifically acetylates the viral coat protein. AtNSI is a nuclear protein but does not act as a transcriptional coactivator in vitro, which distinguishes it from known eukaryotic histone acetyltransferases. Its overexpression enhances the efficiency of infection by Cabbage leaf curl virus. These findings suggest a role for protein acetylation in coordinating replication of the viral DNA genome with its export from the nucleus.