Actomyosin-driven motility and coalescence of phase-separated viral inclusion bodies are required for efficient replication of a plant rhabdovirus.

Phase separation has emerged as a fundamental principle for organizing viral and cellular membraneless organelles. Although these subcellular compartments have been recognized for decades, their biogenesis and mechanisms of regulation are poorly understood. Here, we investigate the formation of membraneless inclusion bodies (IBs) induced during the infection of a plant rhabdovirus, tomato yellow mottle-associated virus (TYMaV). We generated recombinant TYMaV encoding a fluorescently labeled IB constituent protein and employed live-cell imaging to characterize the intracellular dynamics and maturation of viral IBs in infected Nicotiana benthamiana cells. We show that TYMaV IBs are phase-separated biomolecular condensates and that viral nucleoprotein and phosphoprotein are minimally required for IB formation in vivo and in vitro. TYMaV IBs move along the microfilaments, likely through the anchoring of viral phosphoprotein to myosin XIs. Furthermore, pharmacological disruption of microfilaments or inhibition of myosin XI functions suppresses IB motility, resulting in arrested IB growth and inefficient virus replication. Our study establishes phase separation as a process driving the formation of liquid viral factories and emphasizes the role of the cytoskeletal system in regulating the dynamics of condensate maturation.

Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes.

CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop breeding. However, the lack of robust delivery methods has limited the widespread adoption of these revolutionary technologies in plant science. Here, we report an efficient, non-transgenic CRISPR/Cas delivery platform based on the engineered tomato spotted wilt virus (TSWV), an RNA virus with a host range of over 1000 plant species. We eliminated viral elements essential for insect transmission to liberate genome space for accommodating large genetic cargoes without sacrificing the ability to infect plant hosts. The resulting non-insect-transmissible viral vectors enabled effective and stable in planta delivery of Cas12a and Cas9 nucleases as well as adenine and cytosine base editors. In systemically infected plant tissues, the deconstructed TSWV-derived vectors induced efficient somatic gene mutations and base conversions in multiple crop species with little genotype dependency. Plants with heritable, bi-allelic mutations could be readily regenerated by culturing the virus-infected tissues in vitro without antibiotic selection. Moreover, we showed that antiviral treatment with ribavirin during tissue culture cleared the viral vectors in 100% of regenerated plants and further augmented the recovery of heritable mutations. Because many plants are recalcitrant to stable transformation, the viral delivery system developed in this work provides a promising tool to overcome gene delivery bottlenecks for genome editing in various crop species and elite varieties.

Quantification of plasmodesmata frequency under three-dimensional view using focused ion beam-scanning electron microscopy and image analysis.

The quantitative study of plasmodesmata (PD) frequency is routine in plant science for providing information on the potential of intercellular transportation. Here, we report quantification of plasmodesmatal frequency in virus-infected tobacco vascular tissues using serial sectioning and image analysis. The image datasets were collected by focused ion beam-scanning electron microscopy (FIB-SEM), and the measurements of plasmodesmatal frequency were performed after image analysis with commercial computational programs. With a 5-nm step size (less than half the diameter of PD) during FIB sectioning, exhaustive PD sampling was performed in regions of interest. Segmentation of cell wall (CW) and PD from the background densities was performed manually, and PD were assigned automatically to individual CW interfaces by image analysis and then quantified. The PD quantification results were used to compare the plamodesmatal frequencies among different CW interfaces of individual cells and the average frequencies among different cell types were calculated. CWs lacking PD distribution were found in several cellular types, and the PD frequency were used to determine the possible pathways of PD-based symplasmic transportation. The method enables imaging of samples of several cells containing multiple CW interfaces and minimizes PD omission during sectioning and imaging.

Hypoxia-responsive micelles self-assembled from amphiphilic block copolymers for the controlled release of anticancer drugs.

Amphiphilic block copolymers poly(ethylene glycol)-block-poly(methacrylic acid-co-2-nitroimidazole methacrylate) (PEG-b-P(MAA-co-NIMA)) were synthesized by the combination of atom transfer radical polymerization (ATRP), hydrolysis and EDC reactions. These copolymers could self-assemble into spherical micelles in water. 2-Nitroimidazole (NI) groups presented hypoxia-responsive properties under hypoxia conditions. The hydrophobic NI groups could be converted into hydrophilic aminoimidazole (AI) groups, which would lead to the expansion of micelles. Moreover, the content of NI groups in the copolymers would affect the hydrophilic-hydrophobic balance and therefore influence the self-assembly behaviour of the copolymer and the morphologies of the micelles. The copolymer micelles were used as a drug delivery system for controlled release of anticancer drug doxorubicin (DOX). The in vitro cytotoxicity investigation revealed that the DOX-loaded micelles showed higher toxicity to hypoxic cells than to normoxic cells. As a result, the block copolymers are expected to be used as an intelligent carrier for hydrophobic drugs to treat hypoxia-associated diseases.