Viperin-like proteins interfere with RNA viruses in plants.

Plant viruses cause substantial losses in crop yield and quality; therefore, devising new, robust strategies to counter viral infections has important implications for agriculture. Virus inhibitory protein endoplasmic reticulum-associated interferon-inducible (Viperin) proteins are conserved antiviral proteins. Here, we identified a set of Viperin and Viperin-like proteins from multiple species and tested whether they could interfere with RNA viruses in planta. Our data from transient and stable overexpression of these proteins in Nicotiana benthamiana reveal varying levels of interference against the RNA viruses tobacco mosaic virus (TMV), turnip mosaic virus (TuMV), and potato virus x (PVX). Harnessing the potential of these proteins represents a novel avenue in plant antiviral approaches, offering a broader and more effective spectrum for application in plant biotechnology and agriculture. Identifying these proteins opens new avenues for engineering a broad range of resistance to protect crop plants against viral pathogens.

High-efficiency retron-mediated single-stranded DNA production in plants.

Retrons are a class of retroelements that produce multicopy single-stranded DNA (ssDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the overproduction of ssDNA, genome engineering and directed evolution in bacteria, yeast and mammalian cells. Retron-mediated ssDNA production in plants could unlock their potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Here, we demonstrated retron-mediated overproduction of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated clustered regularly interspaced short palindromic repeats-retron-coupled ssDNA overproduction and targeted HDR in N. benthamiana. Overall, we present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications. Graphical Abstract.

Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice.

Precise genome editing by systems such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) requires high-efficiency homology-directed repair (HDR). Different technologies have been developed to improve HDR but with limited success. Here, we generated a fusion between the Cas9 endonuclease and the Agrobacterium VirD2 relaxase (Cas9-VirD2). This chimeric protein combines the functions of Cas9, which produces targeted and specific DNA double-strand breaks (DSBs), and the VirD2 relaxase, which brings the repair template in close proximity to the DSBs, to facilitate HDR. We successfully employed our Cas9-VirD2 system for precise ACETOLACTATE SYNTHASE (OsALS) allele modification to generate herbicide-resistant rice (Oryza sativa) plants, CAROTENOID CLEAVAGE DIOXYGENASE-7 (OsCCD7) to engineer plant architecture, and generate in-frame fusions with the HA epitope at HISTONE DEACETYLASE (OsHDT) locus. The Cas9-VirD2 system expands our ability to improve agriculturally important traits in crops and opens new possibilities for precision genome engineering across diverse eukaryotic species.

Genome Editing Technologies for Rice Improvement: Progress, Prospects, and Safety Concerns.

Rice (Oryza sativa) is an important staple food crop worldwide; to meet the growing nutritional requirements of the increasing population in the face of climate change, qualitative and quantitative traits of rice need to be improved. Stress-tolerant crop varieties must be developed with stable or higher yields under stress conditions. Genome editing and speed breeding have improved the accuracy and pace of rice breeding. New breeding technologies including genome editing have been established in rice, expanding the potential for crop improvement. Recently, other genome editing techniques such as CRISPR-directed evolution, CRISPR-Cas12a, and base editors have also been used for efficient genome editing in rice. Since rice is an excellent model system for functional studies due to its small genome and close syntenic relationships with other cereal crops, new genome-editing technologies continue to be developed for use in rice. In this review, we focus on genome-editing tools for rice improvement to address current challenges and provide examples of genome editing in rice. We also shed light on expanding the scope of genome editing and systems for delivering homology-directed repair templates. Finally, we discuss safety concerns and methods for obtaining transgene-free crops.